Methods of Detecting Polyubiquitin Using Anti-Polyubiquitin Antibodies

ABSTRACT

The invention provides anti-polyubiquitin antibodies and methods of using the same.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.16/914,996, filed Jun. 29, 2020, which is a divisional of U.S.application Ser. No. 15/071,422, filed Mar. 16, 2016, now U.S. Pat. No.10,738,106, which is a divisional of U.S. application Ser. No.13/567,919, filed Aug. 6, 2012, now U.S. Pat. No. 9,321,844, whichclaims priority to U.S. Provisional Application No. 61/515,729, filedAug. 5, 2011, the contents of all of which are hereby incorporated byreference in their entirety for all purposes.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in XML format and is hereby incorporated byreference in its entirety. Said XML copy, created on Apr. 18, 2023, isnamed “2023-04-18_01146-0015-03US_ST26” and is 510484 bytes in size.

FIELD OF THE INVENTION

The present invention relates to the field of anti-polyubiquitinantibodies, and more particularly to anti-polyubiquitin antibodies thatdo not specifically bind to monoubiquitin and that are specific forlinear polyubiquitin and methods of using the same.

BACKGROUND

Ubiquitin is a small protein that has important regulatory roles in awide variety of cellular pathways. The best known of these isubiquitin's role in protein degradation, where covalent attachment ofubiquitin to a target protein enables that targeted protein to berecognized and destroyed by the 26S proteasome (see Wilkinson, Semin.Cell Devel. Biol. 11(3): 141-148 (2000)). The covalent attachment ofubiquitin, a 76 amino acid protein, to a target protein is a three-stepenzymatic process (Pickart, Annu. Rev. Biochem. 70: 503-533 (2001)).First, ubiquitin-activating enzyme E1 forms an ubiquitin-E1 thioester inan ATP-dependent reaction. The ubiquitin is transferred from theubiquitin-E1 thioester to a member of the ubiquitin-conjugating enzyme(E2) family in the second step. In the third step, with the assistanceof a ubiquitin-protein ligase (E3), an isopeptide bond is formed betweenthe carboxyl terminus of ubiquitin and the ε-amino group of a lysineresidue on the target protein. Enzymes termed deubiquitinases removeubiquitin moieties from target proteins (Guterman and Glickman, Curr.Prot. Pep. Sci. 5: 201-210 (2004)).

Ubiquitin contains seven lysine residues (Lys6, Lys11, Lys27, Lys33,Lys29, Lys48, and Lys63), and thus ubiquitin itself may serve as atarget protein for ubiquitination (Peng et al., Nat. Biotechnol. 21:921-926 (2003); Pickart and Fushman, Curr. Opin. Chem. Biol. 8:610-616(2004)). The molecule produced upon ubiquitination of a ubiquitinprotein is termed a polyubiquitin molecule, and may comprise two or moreubiquitin moieties. Ubiquitination of ubiquitin may theoretically occurat any of the seven lysine residues (Peng et al., Nat. Biotechnol. 21:921-926 (2003)), so that different species of polyubiquitins existhaving isopeptide bonds to different lysine residues within ubiquitin.Polyubiquitin chains with internal isopeptide linkages at all sevenlysine resides have been reported. Iwai and Tokunaga, EMBO Reports10:706-713 (2009).

Recently it was discovered that linear polyubiquitin chains also form inwhich the C-terminal glycine of ubiquitin is conjugated to the α-aminogroup of the N-terminal methionine of another ubiquitin molecule. Iwaiand Tokunaga, EMBO Reports 10:706-713 (2009). Linear polyubiquitin isformed via the linear ubiquitin chain assembly complex (LUBAC) which iscomposed of two ring finger proteins, HOIL-1L and HOIP. Tokunaga et al.,Nat. Cell Biol. 11:123-132 (2009). It is believed that geneticallyencoded, unanchored linear polyubiquitin does not exist in cells as itsC-terminus is vulnerable to cleavage by isopeptidase T. Iwai andTokunaga, EMBO Reports 10:706-713 (2009). This observation suggests thatlinear polyubiquitin is assembled onto a substrate proteinpost-translationally and that conjugated linear polyubiquitin moleculesare potential modulators of protein activity and function. Id. Forexample, linear polyubiquitination of the NF-kB essential modulator(NEMO) has been shown to play a role in NF-κB activation. Id.

Antibodies which distinguish linear polyubiquitin over polyubiquitin ofdifferent lysine linkages would be useful to further examine the role oflinear polyubiquitin chains in protein degradation and regulation and totarget and modulate linear polyubiquitin in linearpolyubiquitin-mediated pathways.

SUMMARY

The invention provides anti-linear polyubiquitin antibodies and methodsof using the same. In one embodiment, the invention provides an isolatedantibody that specifically binds a first polyubiquitin comprising aC-terminal to N-terminal linkage, wherein the antibody does notspecifically bind a second polyubiquitin comprising a lysine linkage. Inanother embodiment, the invention provides an isolated antibody thatspecifically binds both a first polyubiquitin comprising a C-terminal toN-terminal linkage and a second polyubiquitin comprising a lysinelinkage, wherein the antibody does not specifically bind monoubiquitin,and wherein the antibody binds the second polyubiquitin with asubstantially reduced binding affinity as compared to the bindingaffinity of the antibody for the first polyubiquitin.

In another embodiment the invention provides an isolated antibody thatspecifically binds C- to N-terminal linked polyubiquitin, wherein theantibody does not specifically bind monoubiquitin. In one aspect, theantibody comprises at least one hypervariable (HVR) sequence selectedfrom HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2, and HVR-H3 of any of SEQ IDNOs: 1, 4, 19 and 50-57; SEQ ID NOs: 2 and 58-63; SEQ ID NOs: 3, 5, 6,20, 21 and 64-72; SEQ ID NOs: 7, 10, 13, 16, 22 and 73-81; SEQ ID NOs:8, 11, 14, 17, 23, 24 and 82-86; and SEQ ID NOs: 9, 12, 15, 18 and87-93, respectively.

In another aspect, the antibody comprises at least one sequence selectedfrom HVR-L1, HVR-L2 and HVR-L3, wherein HVR-L1 comprises the amino acidsequence RASQX₁VX2X₃X₄VA (SEQ ID NO: 39), wherein amino acid X₁ isselected from amino acid D, S and G, amino acid X₂ is selected from Sand D, amino acid X₃ is selected from S, T and N and amino acid X₄ isselected from A and S; wherein HVR-L2 comprises the amino acid sequenceof SEQ ID NO: 2; and wherein HVR-L3 comprises the amino acid sequenceQQX₅X₆X₇X₈X₉PX₁₀T (SEQ ID NO: 40), wherein amino acid X₅ is selectedfrom S, Y and H, amino acid X₆ is selected from Y and F, amino acid X₇is selected from T, Y and A, amino acid X₈ is selected from T, Y and S,amino acid X₉ is optional and if present is serine, and amino acid X₁₀is selected from P and L.

In another aspect, the antibody comprises at least one hypervariable(HVR) sequence selected from HVR-H1, HVR-H2, and HVR-H3 wherein HVR-H1comprises the amino acid sequence X₁₁X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈ (SEQ ID NO:41), wherein amino acid X₁₁ is selected from T and N, amino acid X₁₂ isselected from F and I, amino acid X₁₃ is selected from S, T and Y, aminoacid X₁₄ is selected from N, D, S and Y, amino acid X₁₅ is selected fromT, Y, S and D, amino acid X₁₆ is selected from Y, D and S, amino acidX₁₇ is selected from I and M, and amino acid X₁₈ is selected from S andH; and wherein HVR-H2 comprises the amino acid sequence AX₁₉IX₂₀X₂₁X₂₂X₂₃X₂₄X₂₅TX₂₆ (SEQ ID NO: 42), wherein amino acid X₁₉ isselected from S, G, W and E, amino acid X₂₀ is selected from T, S and Y,amino acid X₂₁ is selected from P and S, amino acid X₂₂ is selected fromS and Y, amino acid X₂₃ is selected from G, S and Y, amino acid X₂₄ isselected from G and S, amino acid X₂₅ is selected from S and Y, andamino acid X₂₆ is selected from D and S, and HVR-H3 comprises the aminoacid sequence RX₂₇X₂₈X₂₉X₃₀X₃₁X₃₂X₃₃X₃₄X₃₅X₃₆X₃₇D (SEQ ID NO: 43)wherein amino acid X₂₇ is selected from T, E and G, amino acid X₂₈ isselected from W, A and Y, amino acid X₂₉ is selected from L, G, V and S,amino acid X₃₀ is selected from L, S and W, amino acid X₃₁ is selectedfrom R, K and Y, amino acid X₃₂ is selected from W, L, G and Y, aminoacid X₃₃ is selected from V, L, A and G, amino acid X₃₇ is selected fromM and F, and wherein amino acids X₃₄, X₃₅, and X₃₆ are optionallypresent and if present, amino acid X₃₄ is S, amino acid X₃₅ is selectedfrom V and P, and amino acid X₃₆ is A.

In another aspect, the antibody comprises at least one hypervariable(HVR) sequence selected from HVR-L1, HVR-L2, and HVR-L3, wherein HVR-L1comprises the amino acid sequence RASQ X₃₈X₃₉X₄₀X₄₁X₄₂X₄₃A (SEQ ID NO:44), wherein amino acid X₃₈ is selected from D, A, E, G, L, N, S, T andV, amino acid X₃₉ is selected from V, A, L and S, amino acid X₄₀ isselected from S, F, G, L, R and V, amino acid X₄₁ is selected from T, G,I, N, S and V, amino acid X₄₂ is selected from A, H, Q, R, S, and Y, andamino acid X₄₃ is selected from V and L, and wherein HVR-L2 comprisesthe amino acid sequence SX₄₄X₄₅X₄₆X₄₇YX₄₈ (SEQ ID NO: 45), wherein aminoacid X₄₄ is selected from A and R, amino acid X₄₅ is selected from S, K,Q, and R, amino acid X₄₆ is selected from F and Y, amino acid X₄₇ isselected from L, A, F, G, H, I, K, M, N, P, R, S, V and Y, and aminoacid X₄₈ is selected from S, A, D, F, G, H, V, W and Y and whereinHVR-L3 comprises the sequence QQ X₄₉X₅₀X₅₁X₅₂PPT (SEQ ID NO: 46),wherein amino acid X₄₉ is selected from H and S, amino acid X₅₀ isselected from Y, K, N, Q, R, S, V, and W, amino acid X₅₁ is selectedfrom T, I, Q, R, S and V, and amino acid X₅₂ is selected from T, A, D,F, G, K, N, P, Q, R, S and V.

In another aspect, the antibody comprises at least one hypervariable(HVR) sequence selected from HVR-H1, HVR-H2 and HVR-H3, wherein HVR-H1comprises the amino acid sequence X₅₃X₅₄X₅₅YX₅₆S (SEQ ID NO: 47),wherein amino acid X₅₃ is selected from A, F, K, M, Q, R and S, aminoacid X₅₄ is selected from N and W, amino acid X₅₅ is selected from T, A,I, L, M, and V, and amino acid X₅₆ is selected from I, M and V, andwherein HVR-L2 comprises the amino acid sequence AX₅₇X₅₈TPX₅₉SGX₆₀TX₆₁(SEQ ID NO:48), wherein amino acid X₅₇ is selected from T and S, aminoacid X₅₈ is selected from I, S and V, amino acid X₅₉ is selected from Sand A, amino acid X₆₀ is selected from S, H, I, L, M and Q, amino acidX₆₁ is selected from D and N, and wherein HVR-H3 comprises the aminoacid sequence X₆₂WX₆₃X₆₄RWVX₆₅D (SEQ ID NO:49) wherein amino acid X₆₂ isselected from S and T, amino acid X₆₃ is selected from L and Y, aminoacid X₆₄ is selected from L, I and V, amino acid X₆₅ is selected from Mand F.

In another aspect, the antibody comprises an HVR-L1 sequence of SEQ IDNO: 1 or 4, an HVR-L2 sequence of SEQ ID NO: 2, and an HVR-L3 sequenceselected from SEQ ID NO: 3, 5 and 6, respectively. In another aspect,the antibody comprises an HVR-H1 sequence selected from SEQ ID NO: 7,10, 13 and 16, an HVR-H2 sequence selected from SEQ ID NO: 11, 23 and24, and an HVR-H3 sequence of SEQ ID NO: 12, respectively. In anotheraspect, the antibody comprises an HVR-L1 sequence selected from SEQ IDNO: 1 and 50-56, an HVR-L2 sequence selected from SEQ ID NO: 2 and 57-62and an HVR-L3 sequence selected from SEQ ID NO: 3 and 63-71,respectively. In another aspect, the antibody comprises an HVR-H1sequence selected from SEQ ID NO: 7 and 72-80, an HVR-H2 sequenceselected from SEQ ID NO: 8 and 81-85, and an HVR-H3 sequence selectedfrom SEQ ID NO: 9 and 86-92, respectively.

In another aspect, the antibody comprises HVR-L1, HVR-L2, and HVR-L3sequences corresponding to those set forth for clones 1E2, 1D8, 1F4 or1A10 in FIG. 1A. In another aspect, the antibody comprises HVR-H1,HVR-H2, and HVR-H3 sequences corresponding to those set forth for clones1E2, 1D8, 1F4 or 1A10 in FIG. 1B. In another aspect, the antibodycomprises HVR-L1, HVR-L2, and HVR-L3 sequences corresponding to thoseset forth for clones 1D8.3C2, 1D8.3F8 or 1D8.4F5 in FIG. 4A. In anotheraspect, the antibody comprises HVR-H1, HVR-H2, and HVR-H3 sequencescorresponding to those set forth for clones 1D8.3C2, 1D8.3F8 or 1D8.4F5in FIG. 4B.

In another aspect, the antibody comprises the HVR-L1 sequence of SEQ IDNO: 1, the HVR-L2 sequence of SEQ ID NO: 2, the HVR-L3 sequence of SEQID NO: 3, the HVR-H1 sequence of SEQ ID NO: 10, the HVR-H2 sequence ofSEQ ID NO: 11 and the HVR-H3 sequence of SEQ ID NO: 12. In anotheraspect, the antibody comprises the HVR-L1 sequence of SEQ ID NO: 19, theHVR-L2 sequence of SEQ ID NO: 2, the HVR-L3 sequence of SEQ ID NO: 3,the HVR-H1 sequence of SEQ ID NO: 10, the HVR-H2 sequence of SEQ ID NO:23 and the HVR-H3 sequence of SEQ ID NO: 12. In another aspect, theantibody comprises the HVR-L1 sequence of SEQ ID NO: 1, the HVR-L2sequence of SEQ ID NO: 2, the HVR-L3 sequence of SEQ ID NO: 20, theHVR-H1 sequence of SEQ ID NO: 10, the HVR-H2 sequence of SEQ ID NO: 11and the HVR-H3 sequence of SEQ ID NO: 12. In another aspect, theantibody comprises the HVR-L1 sequence of SEQ ID NO: 1, the HVR-L2sequence of SEQ ID NO: 2, the HVR-L3 sequence of SEQ ID NO: 20, theHVR-H1 sequence of SEQ ID NO: 10, the HVR-H2 sequence of SEQ ID NO: 11and the HVR-H3 sequence of SEQ ID NO: 12. In another aspect, theantibody comprises the HVR-L1 sequence of SEQ ID NO: 1, the HVR-L2sequence of SEQ ID NO: 2, the HVR-L3 sequence of SEQ ID NO: 3, theHVR-H1 sequence of SEQ ID NO: 7, the HVR-H2 sequence of SEQ ID NO: 8 andthe HVR-H3 sequence of SEQ ID NO: 9. In another aspect, the antibodycomprises the HVR-L1 sequence of SEQ ID NO: 1, the HVR-L2 sequenceselected from SEQ ID NO: 2, 57 and 59, the HVR-L3 sequence selected fromSEQ ID NO: 3, 64 and 71, the HVR-H1 sequence of SEQ ID NO: 7 or 79, theHVR-H2 sequence of SEQ ID NO: 8 or 81, and the HVR-L3 sequence selectedfrom SEQ ID NO: 9, 86, 88 or 89. In another aspect, the antibodycomprises the HVR-L1 sequence of SEQ ID NO: 1, the HVR-L2 sequence ofSEQ ID NO: 57, the HVR-L3 sequence of SEQ ID NO: 3, the HVR-H1 sequenceof SEQ ID NO: 7, the HVR-H2 sequence of SEQ ID NO: 8 and the HVR-H3sequence of SEQ ID NO: 9. In another aspect, the antibody comprises theHVR-L1 sequence of SEQ ID NO: 1, the HVR-L2 sequence of SEQ ID NO: 2,the HVR-L3 sequence of SEQ ID NO: 3, the HVR-H1 sequence of SEQ ID NO:7, the HVR-H2 sequence of SEQ ID NO: 81 and the HVR-H3 sequence of SEQID NO: 9. In another aspect, the antibody comprises the HVR-L1 sequenceof SEQ ID NO: 1, the HVR-L2 sequence of SEQ ID NO: 57, the HVR-L3sequence of SEQ ID NO: 3, the HVR-H1 sequence of SEQ ID NO: 7, theHVR-H2 sequence of SEQ ID NO: 81 and the HVR-H3 sequence of SEQ ID NO:9. In certain aspects any of the antibodies described herein may includea leucine as the first amino acid after the C-terminal end of HVR-H3(e.g., the amino acid immediately adjacent to the C-terminal end of aHVR-H3, such as 1E3, -TWLLRVMDL (SEQ ID NO:96).

In another aspect, the antibody comprises a light chain amino acidsequence selected from SEQ ID NOs: 25-28, 33-35, 94 and 193-195. Inanother aspect, the antibody comprises a heavy chain amino acid sequenceselected from SEQ ID NOs: 29-31, 36-38, 95 and 196-198.

In another aspect, the antibody comprises light chain and heavy chainamino acid sequences with at least 95% sequence identity to the aminoacid sequences of one of the following combinations of sequences: SEQ IDNOs 25 and 29; SEQ ID NOs: 26 and 30; SEQ ID NOs: 27 and 31; SEQ ID NOs:28 and 32; SEQ ID NOs: 33 and 36; SEQ ID NOs: 34 and 37; SEQ ID NOs: 35and 38; SEQ ID NOs: 95 and 95; SEQ ID NOs: 193 and 196; SEQ ID NOs: 194and 197; and SEQ ID NOs: 195 and 198.

In another embodiment, the invention provides an isolated antibody,wherein the antibody binds to the same antigenic determinant on C- toN-terminal-linked polyubiquitin as any one of the foregoing antibodies,and wherein the antibody does not specifically bind to monoubiquitin. Inanother embodiment, the invention provides an isolated antibody thatcompetes with any one of the foregoing antibodies for binding to C- toN-terminal-linked polyubiquitin, wherein the antibody does notspecifically bind to monoubiquitin. In another embodiment, the inventionprovides any of the foregoing isolated antibodies, wherein the antibodyspecifically binds to a C- to N-terminal-linked polyubiquitinatedprotein. In another embodiment, the invention provides any of theforegoing isolated antibodies, wherein the antibody modulates at leastone polyubiquitin-mediated signaling pathway.

In one general aspect, any of the foregoing antibodies is a monoclonalantibody. In another general aspect, any of the foregoing antibodies isa human antibody. In another general aspect, any of the foregoingantibodies is a humanized antibody. In another general aspect, any ofthe foregoing antibodies is a chimeric antibody. In another generalaspect, any of the foregoing antibodies is an antibody fragment thatbinds C- to N-terminal-linked polyubiquitin.

In another embodiment, the invention provides an isolated nucleic acidencoding any of the foregoing antibodies. In another embodiment, theinvention provides a vector comprising an isolated nucleic acid encodingany of the foregoing antibodies. In another embodiment, the inventionprovides a host cell comprising an isolated nucleic acid encoding any ofthe foregoing antibodies. In another embodiment, the invention providesa host cell comprising a vector comprising an isolated nucleic acidencoding any of the foregoing antibodies.

In another embodiment, the invention provides a method of producing anyof the foregoing antibodies, comprising culturing the above-recited hostcell under conditions wherein the antibody is produced. In one aspect,the method further comprises recovering the antibody from the host cell.In another aspect, the method further comprises purification of theantibody.

In another embodiment, the invention provides an immunoconjugatecomprising any of the foregoing antibodies and a cytotoxic agent. Inanother embodiment, the invention provides a pharmaceutical formulationcomprising any of the foregoing antibodies and a pharmaceuticallyacceptable carrier. In one aspect, the pharmaceutical formulationfurther comprises an additional therapeutic agent. In one such aspect,the additional therapeutic agent is a chemotherapeutic agent.

In another embodiment, the invention provides any of the foregoingantibodies for use as a medicament. In another embodiment, the inventionprovides any of the foregoing antibodies for use in treating acell-cycle-related disease or disorder. In one aspect, thecell-cycle-related disease or disorder is selected from a disease ordisorder associated with aberrantly increased cell cycle progression anda disease or disorder associated with aberrantly decreased cell cycleprogression. In one such aspect, the disease or disorder associated withaberrantly increased cell cycle progression is cancer. In another suchaspect, the disease or disorder associated with aberrantly decreasedcell cycle progression is selected from a degenerative muscle disorderand a degenerative nerve disorder.

In another embodiment, the invention provides the use of any of theforegoing antibodies in the manufacture of a medicament. In one aspect,the medicament is for a disease or disorder selected from cancer, adegenerative muscle disorder, and a degenerative nerve disorder. Inanother embodiment, the invention provides a method of treating anindividual having a disease or disorder selected from cancer, adegenerative muscle disorder, and a degenerative nerve disorder,comprising administering to the individual an effective amount of any ofthe foregoing antibodies.

In another embodiment, the invention provides a method of determiningthe presence of a polyubiquitin or polyubiquitinated protein in a samplesuspected of containing a polyubiquitin or polyubiquitinated protein,comprising exposing the same to at least one of the foregoing antibodiesand determining the binding of the at least one antibody to apolyubiquitin or polyubiquitinated protein in the sample. In anotherembodiment, the invention provides a method of separating C- toN-terminal-linked polyubiquitinated protein from non-C- toN-terminal-linked polyubiquitinated protein in a sample, comprisingcontacting the sample with at least one of the foregoing antibodies. Inanother embodiment, the invention provides a method of determining thefunction and/or activity of C- to N-terminal-linked polyubiquitin in acell or sample comprising contacting the cell or sample with at leastone of the foregoing antibodies and assessing the effect of saidcontacting step on the cell or sample.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the results of a phage spot ELISA demonstrating therelative binding signals at a wavelength of 450 nm for clones 1D8, 1E3,1F4 and 1A10 to a panel of ubiquitin proteins, as described in Example1B. The Fab library clones each contained a gD tag and display of theFab on phage was assessed by binding to an anti-gD antibody. An uncoatedwell was used as a negative control.

FIGS. 2A and 2B depict the light and heavy chain amino acid sequences ofthe Fabs obtained in Example 1. FIG. 2A depicts the light chain sequenceof clones 1E3, 1D8, 1F4 and 1A10 (SEQ ID NOs: 25-28, respectively). FIG.2B depicts the heavy chain sequence alignment of clones 1E3, 1D8, 1F4and 1A10 (SEQ ID NOs: 29-32, respectively). In both FIGS. 2A and 2B, theHVR sequences for each clone are indicated by the boxed regions, withthe first box indicating HVR-L1 (FIG. 2A) or HVR-H1 (FIG. 2B), thesecond box indicating HVR-L2 (FIG. 2A) or HVR-H2 (FIG. 2B), and thethird box indicating HVR-L3 (FIG. 2A) or HVR-H3 (FIG. 2B).

FIG. 3 depicts the results of a phage IC₅₀ competition ELISA to measurethe affinity of the purified 1F4, 1D8 and 1E3 Fabs for lineardiubiquitin.

FIG. 4 shows the results of a western blot analysis to determine theability of the 1D8 and 1E3 Fabs to specifically recognize a panel ofdiubiquitin proteins in an immobilized context.

FIGS. 5A and 5B depict the light and heavy chain amino acid sequences ofthe affinity matured clones obtained in Example 2 from the 1D8 affinitymaturation library sorts. FIG. 5A depicts the light chain sequences ofthe affinity matured clones 1D8.3C2, 1D8.3F8 and 1D8.4F5 (SEQ ID NOs:33-35, respectively). FIG. 5A discloses the 1D8 sequence as SEQ ID NO:26. FIG. 5B depicts the heavy chain sequence alignment of the affinitymatured clones 1D8.3C2, 1D8.3F8 and 1D8.4F5 (SEQ ID NOs: 36-38,respectively). FIG. 5B discloses the 1D8 sequence as SEQ ID NO: 30. Inboth FIGS. 5A and 5B, the HVR sequences for each clone are indicated bythe boxed regions, with the first box indicating HVR-L1 (FIG. 5A) orHVR-H1 (FIG. 5B), the second box indicating HVR-L2 (FIG. 5A) or HVR-H2(FIG. 5B), and the third box indicating HVR-L3 (FIG. 5A) or HVR-H3 (FIG.5B). Amino acid changes relative to the 1D8 parental sequence arehighlighted in gray.

FIG. 6 depicts the results of a phage IC₅₀ competition ELISA to measurethe affinity of the 1D8, 1D8.3C2, 1D8.3F8 and 1D8.4F5 Fabs for lineardiubiquitin.

FIGS. 7A and B depict the results of studies assessing the bindingspecificity characteristics of the mutant affinity matured variants incomparison to the parental 1E3 clone and controls. The binding of theparental 1E3 clone and 11 single mutant affinity matured variantsdisplayed on phage were tested for binding to a panel of ubiquitinproteins in an ELISA. Binding to an anti-gD antibody was used to assessFab display on phage and uncoated wells were used as a negative control.

FIGS. 8A and B depict the results of studies assessing the bindingspecificity characteristics of single mutant and double mutant variantsin comparison to the parental 1E3 clone and controls, as described inExample 3. The binding of the parental 1E3 clone, 4 single mutantaffinity matured variants and 3 double mutant affinity matured variants,displayed on phage, were tested for binding to a panel of ubiquitinproteins in an ELISA. Binding to an anti-gD antibody was used to assessFab display on phage and uncoated wells were used as a negative control.

FIGS. 9A and 9B depict the light and heavy chain amino acid sequences ofthe 1E3 Fab, affinity matured clones obtained in Example 3 from thesecond 1E3 affinity maturation library sorts (1F11 and 3F5), the Y102Lmutation observed in clone 4E4 and the triple mutant Fab incorporatingthe amino acid changes from clones 1F11 and 3F5 and the Y102L mutationas described in Example 3P. FIG. 9A depicts the light chain sequences ofthe affinity matured clones for 1E3, 1F11, 3F5, Y102L and 1F11/3F5/Y102L(SEQ ID NOs: 25, 193, 194, 195 and 94, respectively). FIG. 9B depictsthe heavy chain sequence alignment of the affinity matured clones for1E3, 1F11, 3F5, Y102L and 1F11/3F5/Y102L (SEQ ID NOs: 29, 196, 197, 198and 95, respectively). In both FIGS. 9A and 9B, the Kabat positionstargeted for amino acid variation in the second affinity maturationlibraries for 1E3 are indicated by the boxed regions. Amino acid changesrelative to the 1E3 parental sequence are highlighted in gray.

FIG. 10 provides western blot analyses of binding of parental 1E3,single, and double mutant IgGs to linear diubiquitin (serial dilutionsof 1000, 333, 111, 37 and 12 ng per lane) and K63-linked diubiquitin(1000 ng per lane).

FIG. 11 provides western blot analysis of binding of parental 1E3,double, and triple mutant IgGs to linear diubiquitin (serial dilutionsof 1000, 333, 111, 37 and 12 ng per lane) and K63-linked diubiquitin(1000 ng per lane).

FIG. 12 provides western blot analysis of binding of Y102L, Y1012L T110Amutant and various mutant combinations of Y102L, T110A, 3F5 and 1F11IgGs to linear diubiquitin (serial dilutions of 1000, 333, 111, 37 and12 ng per lane) and K63-linked diubiquitin (1000 ng per lane).

FIG. 13 provides western blot analysis of binding of parental 1E3,single, double, and triple mutant IgGs, as described in Example 3M, tolinear diubiquitin (serial dilutions of 1000, 333, 111, 37 and 12 ng perlane) and K63-linked diubiquitin (1000 ng per lane).

FIG. 14 provides western blot analyses of binding of 1E3 and1F11/3F5/Y102L IgGs to two-fold serial dilutions of linear diubiquitin(1000, 500, 250, 125, 63, 31, and 16 ng/lane where gradient isindicated) or monoubiquitin, K11-linked diubiquitin, K48-linkeddiubiquitin, and K63-linked diubiquitin (1 μg/lane). As a control theanti-K63 IgG, Apu3. A8 was analyzed for binding to two-fold serialdilutions of K63-linked diubiquitin (1000, 500, 250, 125, 63, 31, and 16ng/lane where gradient is indicated) or monoubiquitin, lineardiubiquitin, K11-lined diubiquitin, and K48-linked diubiquitin (1μg/lane). The Coomassie stained gel (upper left panel) provides anindication of where each of the tested ubiquitin migrates in the gels.

FIG. 15 depicts the results of experiments in which monoubiquitin,linear polyubiquitin 2-7 (two to seven ubiquitin subunits in length),K48-linked polyubiquitin 2-7 (two to seven ubiquitin subunits inlength), K63-linked polyubiquitin 2-7 (two to seven ubiquitin subunitsin length), and K11-linked polyubiquitin (1 μg each per lane) wereimmunoblotted with a pan-ubiquitin antibody P4D1 (middle panel) or the1F11/3F5/Y102L IgG (right panel). Coomassie staining revealed thecomposition of the samples (left panel). A long and short exposure ofthe western blots is shown.

FIGS. 16A and B depict the results of experiments in which lysates ofHeLa S3 cells treated with varying concentrations of TNFα and forvarying amounts of time with 5.8 μM MG132 were immunoblotted for linearubiquitin chains with the hybrid antibody 1F11/3F5/Y102L or forK63-linked ubiquitin chains with the Apu3. A8 antibody. As a control 250ng each of purified linear polyubiquitin 2-7 and K63-linkedpolyubiquitin 2-7 was run on each gel (FIG. 16A). To assess the level ofNFκB pathway activation the lysates were blotted for IκBα levels (FIG.16B). As a loading control the lysates were blotted for β-tubulin.

FIG. 17 depicts the results of immunoprecipitation experiments with thehybrid anti-linear polyubiquitin antibody 1F11/3F5/Y102L, an isotypecontrol, or an anti-K63 antibody Apu3. A8. The IP experiments wereperformed in 4 M urea IP buffer under three conditions (a mixture of allubiquitin chains, no linear ubiquitin chains, or only linear ubiquitinchains). As a control 1 μg each of purified linear polyubiquitin 2-7 andK63-linked polyubiquitin 2-7 was run on each gel.

FIGS. 18A and B depict the results of immunoprecipitation experimentsusing a 1:1 mixture of linear polyubiquitin 2-7 and K63-linkedpolyubiquitin 2-7 with the anti-linear polyubiquitin antibody1F11/3F5/Y102L or the anti-K63 antibody Apu3. A8 in varyingconcentrations of urea or PBST which were immunoblotted with eitherIF11/3F5/Y102L (anti-linear) or Apu3. A8 (anti-K63). As a control 1 μgeach of purified linear polyubiquitin 2-7 and K63-linked polyubiquitin2-7 was run on each gel. FIG. 18A shows the results of IP experiments inOM, 2M, 4M and 6M urea. FIG. 18B shows the results of IP experiments in6M, 7M, and 8M urea.

FIG. 19 shows the results of immunoprecipitation and immunoblottingexperiments using a 1:1:1:1 mixture of linear polyubiquitin 2-7,K11-linked polyubiquitin, K48-linked polyubiquitin, and K63-linkedpolyubiquitin 2-7 with the anti-linear polyubiquitin antibody1F11/3F5/Y102L, an isotype control, or the anti-K63 polyubiquitinantibody Apu3. A8 crosslinked to Protein A beads in varyingconcentrations of urea. Antibodies used in the immunoblotting were1F11/3F5/Y102L (anti-linear), 2A3/2E6 (anti-K11), Apu2.07 (anti-K48), orApu3. A8 (anti-K63) IgG. As a control 1 μg each of purified linearpolyubiquitin 2-7, K11-linked polyubiquitin, K48-linked polyubiquitin2-7, and K63-linked polyubiquitin 2-7 was run on each gel.

FIG. 20 shows the results of immunoprecipitation and immunoblottingexperiments using a 1:1:1:1 mixture of linear polyubiquitin 2-7,K11-linked polyubiquitin, K48-linked polyubiquitin, and K63-linkedpolyubiquitin 2-7 with the hybrid anti-linear polyubiquitin antibody1F11/3F5/Y102L, an isotype control, or the anti-K63 polyubiquitin IgGantibody Apu3. A8 crosslinked to Protein G beads in varyingconcentrations of urea. Antibodies used in immunoblotting were1F11/3F5/Y102L (anti-linear), 2A3/2E6 (anti-K11), Apu2.07 (anti-K48), orApu3. A8 (anti-K63) IgG. As a control 1 μg each of purified linearpolyubiquitin 2-7, K11-linked polyubiquitin, K48-linked polyubiquitin2-7, and K63-linked polyubiquitin 2-7 was run on each gel.

FIG. 21A depicts the results of immunoprecipitation and immunoblottingexperiments from a 1:1:1:1 mixture of linear polyubiquitin 2-7,K11-linked polyubiquitin, K48-linked polyubiquitin, and K63-linkedpolyubiquitin 2-7 with the anti-linear polyubiquitin antibody1F11/3F5/Y102L, an isotype control, or the anti-K63 polyubiquitinantibody Apu3. A8 in varying concentrations of urea. Antibodies used inimmunoblotting were 1F11/3F5/Y102L (anti-linear), 2A3/2E6 (anti-K11),Apu2.07 (anti-K48), or Apu3. A8 (anti-K63) IgG. As a control 500 ng eachof purified linear polyubiquitin 2-7, K11-linked polyubiquitin,K48-linked polyubiquitin 2-7, and K63-linked polyubiquitin 2-7 was runon each gel.

FIG. 21B depicts the results of immunoprecipitation experiments from a1:1:1:1 mixture of linear polyubiquitin 2-7, K11-linked polyubiquitin,K48-linked polyubiquitin, and K63-linked polyubiquitin 2-7 with thehybrid anti-linear polyubiquitin antibody 1F11/3F5/Y102L, an isotypecontrol, or the anti-K63 polyubiquitin antibody Apu3. A8 in varyingconcentrations of urea and separated by SDS-PAGE gel and Coomassiestained. Regions excised for mass spec AQUA are indicated.

FIG. 21C is a graph which indicates the amount in picomoles ofpolyubiquitin linkages identified by mass spec AQUA from the1F11/3F5/Y102L immunoprecipitations as shown in FIG. 24B (region B andregion C) and described in Example 4D.

FIG. 21D is a graph which indicates the linkage composition ofpolyubiquitin chains identified by mass spec AQUA from the1F11/3F5/Y102L immunoprecipitations as shown in FIG. 24B (region B andregion C).

FIG. 21E is a graph which indicates the percent of linear chainsrecovered in the 1F11/3F5/Y102L immunoprecipitations as identified bymass spec AQUA.

FIG. 22A depicts immunoblots with lysates from 293T cells transfectedwith a plasmid over-expressing Hoil-1L and Hoip or an empty vector. Theblots were probed for linear polyubiquitin, Hoil-1L, Hoip, and β-tubulinas described in Example 4E.

FIG. 22B depicts the results of immunoprecipitation experiments fromlysates of 293T cells transfected with a plasmid over-expressing Hoil-1Land Hoip or an empty vector. Immunoblots were probed for linearpolyubiquitin or Coomassie stained. The indicated regions on theCoomassie-stained gel were excised for mass spec AQUA.

FIG. 22C is a graph which indicates the polyubiquitin linkagecomposition, identified by mass spec AQUA, in the 1F11/3F5/Y102Limmunoprecipitations from Hoil-1L/Hoip over-expressing cells as shown inFIG. 22B.

FIG. 22D depicts the immunofluorescence of HeLa S3 cells transfectedwith a plasmid over-expressing Hoil-1L and Hoip or an empty vector andstained with the anti-linear polyubiquitin antibody 1F11/3F5/Y102L.Addition of recombinant linear polyubiquitin of five or seven subunits(+) competes for binding of the anti-linear polyubiquitin antibody.

FIG. 23 depicts various views of the co-crystal structure of the complexformed between the 1F11/3F5/Y102L Fab fragment and linear diubiquitin.A) 1F11/3F5/Y102L is shown as a cartoon diagram at the bottom of thefigure and linear diubiquitin is depicted as a cartoon diagram insidethe space-filled model at the top. The proximal and distal ubiquitinsubunits and the linear linkage are indicated. B) The epitope on lineardiubiquitin is shown in dark gray on the surface of the diubiquitin thatinteracts with 1F11/3F5/Y102L. Residues which have at least 25% of theirsolvent accessible surface area buried at the interface of lineardiubiquitin and 1F11/3F5/Y102L and/or are within 4.5 Å of the Fab areindicated by single letter amino acid code and residue number. Panel Bof FIG. 23 discloses residues 31-37, 70-76 and 60-63 as SEQ ID NOS379-381, respectively. C) The paratope on 1F11/3F5/Y102L Fab is shown indark gray on the surface of the Fab that interacts with diubiquitin.Residues which have at least 25% of their solvent accessible surfacearea buried at the interface of 1F11/3F5/Y102L and diubiquitin and/orare within 4.5 Å of the diubiquitin are indicated by single letter aminoacid code and residue number. Panel C of FIG. 23 discloses residues95-99 and 52-56 as SEQ ID NOS 378 and 377, respectively.

FIG. 24 shows additional close up views of the co-crystal structure ofthe complex formed between the 1F11/3F5/Y102L Fab fragment and lineardiubiquitin. A) Close up showing the hydrogen bonds formed between theside chain of Gln56 of the heavy chain and the main chain carbonylgroups of Gly75 and Gly76. Diubiquitin is shown in light gray and theFab in dark grey. B) Potential electrostatic interaction between Lys52of the light chain and Asp32 and dipole from the carboxy-terminal end ofthe alpha helix of diubiquitin. Diubiquitin is shown in light gray andthe Fab in dark gray. C) Hydrophobic interactions between Leu102 of theheavy chain and Val2 and Leu4 of framework 1 of the heavy chain. Leu102is at the carboxyl-terminal end of CDR H3. Diubiquitin is shown in lightgray and the Fab in dark gray.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION I. Definitions

An “acceptor human framework” for the purposes herein is a frameworkcomprising the amino acid sequence of a light chain variable domain (VL)framework or a heavy chain variable domain (VH) framework derived from ahuman immunoglobulin framework or a human consensus framework, asdefined below. An acceptor human framework “derived from” a humanimmunoglobulin framework or a human consensus framework may comprise thesame amino acid sequence thereof, or it may contain amino acid sequencechanges. In some embodiments, the number of amino acid changes are 10 orless, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less,3 or less, or 2 or less. In some embodiments, the VL acceptor humanframework is identical in sequence to the VL human immunoglobulinframework sequence or human consensus framework sequence.

“Affinity” refers to the strength of the sum total of noncovalentinteractions between a single binding site of a molecule (e.g., anantibody) and its binding partner (e.g., an antigen). Unless indicatedotherwise, as used herein, “binding affinity” refers to intrinsicbinding affinity which reflects a 1:1 interaction between members of abinding pair (e.g., antibody and antigen). The affinity of a molecule Xfor its partner Y can generally be represented by the dissociationconstant (Kd). Affinity can be measured by common methods known in theart, including those described herein. Specific illustrative andexemplary embodiments for measuring binding affinity are described inthe following.

An “affinity matured” antibody refers to an antibody with one or morealterations in one or more hypervariable regions (HVRs), compared to aparent antibody which does not possess such alterations, suchalterations resulting in an improvement in the affinity of the antibodyfor antigen.

An “agonist antibody” as used herein is an antibody which mimics atleast one of the functional activities of a polypeptide of interest.

An “antagonist antibody” or a “blocking antibody” is an antibody whichinhibits or reduces biological activity of the antigen to which itspecifically binds. Certain blocking antibodies or antagonist antibodiessubstantially or completely inhibit the biological activity of theantigen.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-binding activity.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)₂; diabodies; linear antibodies; single-chain antibody molecules(e.g. scFv); and multispecific antibodies formed from antibodyfragments.

An “antibody that binds to the same epitope” as a reference antibodyrefers to an antibody that blocks binding of the reference antibody toits antigen in a competition assay by 50% or more, and conversely, thereference antibody blocks binding of the antibody to its antigen in acompetition assay by 50% or more. An exemplary competition assay isprovided herein.

The terms “anti-linear linked polyubiquitin antibody” and “an antibodythat binds to linear linked polyubiquitin” refer to an antibody that iscapable of binding linear linked polyubiquitin with sufficient affinitysuch that the antibody is useful as a diagnostic and/or therapeuticagent in targeting linear linked polyubiquitin. In one embodiment, theextent of binding of an anti-linear linked polyubiquitin antibody to anunrelated, non-linear linked polyubiquitin protein is less than about10% of the binding of the antibody to linear linked polyubiquitin asmeasured, e.g., by a radioimmunoassay (RIA). In certain embodiments, anantibody that binds to linear linked polyubiquitin has a dissociationconstant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, K 0.01 nM, or≤0.001 nM (e.g. 10⁻⁸ M or less, e.g. from 10⁻⁸ M to 10⁻¹³ M, e.g., from10⁻⁹ M to 10⁻¹³ M). In certain embodiments, an anti-linear linkedpolyubiquitin antibody binds to an epitope of linear linkedpolyubiquitin that is conserved among linear linked polyubiquitin fromdifferent species.

As used herein, the term “anti-polyubiquitin antibody” refers to anantibody that is capable of specifically binding to a polyubiquitinmolecule.

As used herein, the terms “anti-ubiquitin antibody” and“anti-monoubiquitin antibody” are used interchangeably, and refer to anantibody that is capable of specifically binding to a ubiquitinmolecule.

The term “chimeric” antibody refers to an antibody in which a portion ofthe heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species.

The “class” of an antibody refers to the type of constant domain orconstant region possessed by its heavy chain. There are five majorclasses of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of thesemay be further divided into subclasses (isotypes), e.g., IgG₁, IgG₂,IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constant domains thatcorrespond to the different classes of immunoglobulins are called α, δ,ε, γ, and μ, respectively.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents a cellular function and/or causes cell death ordestruction. Cytotoxic agents include, but are not limited to,radioactive isotopes (e.g., At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³,Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu); chemotherapeuticagents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids(vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycinC, chlorambucil, daunorubicin or other intercalating agents); growthinhibitory agents; enzymes and fragments thereof such as nucleolyticenzymes; antibiotics; toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof; and the variousantitumor or anticancer agents disclosed below.

A “disorder” is any condition that would benefit from treatment with anantibody of the invention. This includes chronic and acute disorders ordiseases including those pathological conditions which predispose themammal to the disorder in question. Non-limiting examples of disordersto be treated herein include cancer, and hypotrophy disorders including,but not limited to, degenerative muscle disorders and degenerative nervedisorders.

“Effector functions” refer to those biological activities attributableto the Fc region of an antibody, which vary with the antibody isotype.Examples of antibody effector functions include: C1q binding andcomplement dependent cytotoxicity (CDC); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g. B cell receptor); and B cellactivation.

An “effective amount” of an agent, e.g., a pharmaceutical formulation,refers to an amount effective, at dosages and for periods of timenecessary, to achieve the desired therapeutic or prophylactic result.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain that contains at least a portion of theconstant region. The term includes native sequence Fc regions andvariant Fc regions. In one embodiment, a human IgG heavy chain Fc regionextends from Cys226, or from Pro230, to the carboxyl-terminus of theheavy chain. However, the C-terminal lysine (Lys447) of the Fc regionmay or may not be present. Unless otherwise specified herein, numberingof amino acid residues in the Fc region or constant region is accordingto the EU numbering system, also called the EU index, as described inKabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, MD,1991.

“Framework” or “FR” refers to variable domain residues other thanhypervariable region (HVR) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingsequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The terms “full length antibody,” “intact antibody,” and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native antibody structure orhaving heavy chains that contain an Fc region as defined herein.

The terms “host cell,” “host cell line,” and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues.

A “human consensus framework” is a framework which represents the mostcommonly occurring amino acid residues in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat et al., Sequences of Proteins of ImmunologicalInterest, FifthEdition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In oneembodiment, for the VL, the subgroup is subgroup kappa I as in Kabat etal., supra. In one embodiment, for the VH, the subgroup is subgroup IIIas in Kabat et al., supra.

A “humanized” antibody refers to a chimeric antibody comprising aminoacid residues from non-human HVRs and amino acid residues from humanFRs. In certain embodiments, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the HVRs (e.g., CDRs) correspond tothose of a non-human antibody, and all or substantially all of the FRscorrespond to those of a human antibody. A humanized antibody optionallymay comprise at least a portion of an antibody constant region derivedfrom a human antibody. A “humanized form” of an antibody, e.g., anon-human antibody, refers to an antibody that has undergonehumanization.

The term “hypervariable region” or “HVR,” as used herein, refers to eachof the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops (“hypervariable loops”).Generally, native four-chain antibodies comprise six HVRs; three in theVH (H1, H2, H3), and three in the VL (L1, L2, L3). HVRs generallycomprise amino acid residues from the hypervariable loops and/or fromthe “complementarity determining regions” (CDRs), the latter being ofhighest sequence variability and/or involved in antigen recognition.Exemplary hypervariable loops occur at amino acid residues 26-32 (L1),50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3).(Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987).) Exemplary CDRs(CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) occur at amino acidresidues 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 ofH2, and 95-102 of H3. (Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, MD (1991).) With the exception of CDR1in VH, CDRs generally comprise the amino acid residues that form thehypervariable loops. CDRs also comprise “specificity determiningresidues,” or “SDRs,” which are residues that contact antigen. SDRs arecontained within regions of the CDRs called abbreviated-CDRs, or a-CDRs.Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, anda-CDR-H3) occur at amino acid residues 31-34 of L1, 50-55 of L2, 89-96of L3, 31-35B of H1, 50-58 of H2, and 95-102 of H3. (See Almagro andFransson, Front. Biosci. 13:1619-1633 (2008).) Unless otherwiseindicated, HVR residues and other residues in the variable domain (e.g.,FR residues) are numbered herein according to Kabat et al., supra.

An “immunoconjugate” is an antibody conjugated to one or moreheterologous molecule(s), including but not limited to a cytotoxicagent.

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g., cows, sheep, cats, dogs, andhorses), primates (e.g., humans and non-human primates such as monkeys),rabbits, and rodents (e.g., mice and rats). In certain embodiments, theindividual or subject is a human.

An “isolated” antibody is one which has been separated from a componentof its natural environment. In some embodiments, an antibody is purifiedto greater than 95% or 99% purity as determined by, for example,electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillaryelectrophoresis) or chromatographic (e.g., ion exchange or reverse phaseHPLC). For review of methods for assessment of antibody purity, see,e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

An “isolated” nucleic acid refers to a nucleic acid molecule that hasbeen separated from a component of its natural environment. An isolatednucleic acid includes a nucleic acid molecule contained in cells thatordinarily contain the nucleic acid molecule, but the nucleic acidmolecule is present extrachromosomally or at a chromosomal location thatis different from its natural chromosomal location.

“Isolated nucleic acid encoding an anti-linear linked polyubiquitinantibody” refers to one or more nucleic acid molecules encoding antibodyheavy and light chains (or fragments thereof), including such nucleicacid molecule(s) in a single vector or separate vectors, and suchnucleic acid molecule(s) present at one or more locations in a hostcell.

As used herein, the terms “linear linked polyubiquitin” and “C-terminalto N-terminal linked polyubiquitin” are interchangeable, and refer to apolyubiquitin molecule comprising at least one isopeptide bond betweenthe C-terminus (e.g., the C-terminal glycine) of one ubiquitin moietyand the N-terminal α-amino group (e.g., the N-terminal methionine) ofanother ubiquitin moiety.

As used herein, “lysine linkage” indicates a linkage between oneubiquitin moiety and another ubiquitin moiety which involves a lysineresidue (e.g., K6, K11, K27, K29, K33, K48, and/or K63).

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g., containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen. Thus, the modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies to be used in accordance with the presentinvention may be made by a variety of techniques, including but notlimited to the hybridoma method, recombinant DNA methods, phage-displaymethods, and methods utilizing transgenic animals containing all or partof the human immunoglobulin loci, such methods and other exemplarymethods for making monoclonal antibodies being described herein.

A “naked antibody” refers to an antibody that is not conjugated to aheterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The nakedantibody may be present in a pharmaceutical formulation.

“Native antibodies” refer to naturally occurring immunoglobulinmolecules with varying structures. For example, native IgG antibodiesare heterotetrameric glycoproteins of about 150,000 daltons, composed oftwo identical light chains and two identical heavy chains that aredisulfide-bonded. From N- to C-terminus, each heavy chain has a variableregion (VH), also called a variable heavy domain or a heavy chainvariable domain, followed by three constant domains (CH1, CH2, and CH3).Similarly, from N- to C-terminus, each light chain has a variable region(VL), also called a variable light domain or a light chain variabledomain, followed by a constant light (CL) domain. The light chain of anantibody may be assigned to one of two types, called kappa (κ) andlambda (λ), based on the amino acid sequence of its constant domain.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco,California, or may be compiled from the source code. The ALIGN-2 programshould be compiled for use on a UNIX operating system, including digitalUNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2program and do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical formulation, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

As used herein, the term “polyubiquitin” is defined as all species ofnative human and synthetic polymeric chains of ubiquitin which fallwithin human and synthetic classes of different polymeric linkages ofubiquitin, including, but not limited to, linear polyubiquitin,K6-linked polyubiquitin, K11-linked polyubiquitin, K27-linkedpolyubiquitin, K29-linked polyubiquitin, K33-linked polyubiquitin,K48-linked polyubiquitin and K63-linked polyubiquitin. Polyubiquitin maybe of any length, and includes at least two ubiquitin moieties.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of the individual being treated, and can beperformed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include, but are not limitedto, preventing occurrence or recurrence of disease, alleviation ofsymptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, preventing metastasis, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis. In some embodiments, antibodies ofthe invention are used to delay development of a disease or to slow theprogression of a disease.

As used herein, the terms “ubiquitin” and “monoubiquitin” are usedinterchangeably, and refer to any native ubiquitin from any vertebratesource, including mammals such as primates (e.g. humans) and rodents(e.g., mice and rats), unless otherwise indicated. The term encompasses“full-length,” unprocessed ubiquitin as well as any shortened orposttranslationally modified form of ubiquitin that results fromprocessing in the cell, excepting molecules comprised of multipleubiquitin moieties. The term also encompasses naturally occurringvariants of ubiquitin, e.g., splice variants or allelic variants. Theamino acid sequence of an exemplary human ubiquitin is shown in SEQ IDNO:97: MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGG (SEQ ID NO: 97). Ubiquitin has at leastone lysine residue at amino acid 6, amino acid 11, amino acid 27, aminoacid 29, amino acid 33, amino acid 48, and/or amino acid 63 (marked inbold in SEQ ID NO: 97, above).

As used herein, the term “ubiquitin pathway” refers to a biochemicalpathway in a cell or reconstituted in vitro that includes ubiquitinand/or polyubiquitin.

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (VH and VL, respectively) of a native antibody generally havesimilar structures, with each domain comprising four conserved frameworkregions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindtet al. Kuby Immunology, 6^(th) ed., W.H. Freeman and Co., page 91(2007).) A single VH or VL domain may be sufficient to conferantigen-binding specificity. Furthermore, antibodies that bind aparticular antigen may be isolated using a VH or VL domain from anantibody that binds the antigen to screen a library of complementary VLor VH domains, respectively. See, e.g., Portolano et al., J. Immunol.150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

The term “vector,” as used herein, refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked. Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. Certain vectors are capable of directingthe expression of nucleic acids to which they are operatively linked.Such vectors are referred to herein as “expression vectors.”

II. Compositions and Methods

In one aspect, the invention is based, in part, on the creation ofantibodies that are capable of specifically recognizing a firstpolyubiquitin molecule containing a first polyubiquitin linkage but notspecifically binding to a second polyubiquitin molecule containing asecond polyubiquitin linkage. In certain embodiments, antibodies thatspecifically bind to linear polyubiquitin or C-terminal toN-terminal-linked polyubiquitin are provided. Antibodies of theinvention are useful both in research and, e.g., for the diagnosis ortreatment, e.g., of diseases and disorders relating to aberrant cellcycle progression.

The unique properties of the anti-linear polyubiquitin antibodies of theinvention make them particularly useful for distinguishing betweendifferent linked forms of polyubiquitin in a cellular system withoutresorting to cumbersome and expensive genetic manipulation orbiophysical methods such as mass spectrometry. The anti-linear linkedpolyubiquitin antibodies of the invention can be used to characterizethe function(s) and activities of specific linear polyubiquitins both invitro and in vivo. The anti-linear linked polyubiquitin antibodies ofthe invention can also be used to determine the role of specific linearpolyubiquitins in the development and pathogenesis of disease. Theanti-linear linked polyubiquitin antibodies of the invention can furtherbe used to treat diseases in which one or more specific linearpolyubiquitins are aberrantly regulated or aberrantly functioningwithout interfering with the normal activity of polyubiquitins for whichthe anti-polyubiquitin antibodies are not specific.

The involvement of the ubiquitin system in the NFκ-B pathway has beendescribed. Karin et al., Nature Rev. Immunol. 5:749-759 (2005) and Chen,Z. J., Nature Cel Biol. 7:758-765 (2005). Stimulation of cells withproinflammatory cytokines has been shown to result in K63-linkedpolyubiquitination of RIP1 and NEMO proteins which result in activationof IκB kinase (IKK). Tokunaga et al., Nature Cell Biol. 11:123-132(2009). IKK is then polyubiquitinated with K48-linked polyubiquitin andsubsequently degraded, leading to activation of NFκ-B. Id. Recently itwas also shown that LUBAC activates the NFκ-B pathway but not the INKpathway via linear polyubiquitination of NFκ-B. Id. NF-κB also plays arole in cell proliferation and the cell cycle. Many cancers displayaberrant activation of NF-κB and suppression of NF-κB suppresses cancercell proliferation. Garg and Aggarwal, Leukemia 16: 1053-68 (2002). Thepresence of linear-linked polyubiquitin on proteins such as NF-κB playsan important role in modulation of NF-κB activity and cell cycleprogression. Thus, the antibodies and Fabs of the invention provide auseful therapeutic means for modulation of disorders and disease statesin which cell cycle regulation is aberrant. In one embodiment, theanti-linear polyubiquitin antibodies of the invention are used to treatdiseases and disorders where cell cycle progression is aberrantlyupregulated, resulting in too much cell division, such as cancer. Inanother embodiment, the anti-linear-linked polyubiquitin antibodies ofthe invention are used to treat diseases and disorders where cell cycleprogression is aberrantly downregulated, resulting in too little celldivision and concomitant wasting or destruction of tissue. Examples ofsuch diseases include, but are not limited to, degenerative muscledisorders and degenerative nerve disorders (including, but not limitedto, Charcot Marie Tooth syndrome, poliomyelitis, amyotrophic lateralsclerosis, and Guillain-Barre syndrome).

As used herein, the terms “cell proliferative disorder” and“proliferative disorder” refer to disorders that are associated withsome degree of abnormal cell proliferation. In one embodiment, the cellproliferative disorder is cancer.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth/proliferation. Examples of cancer include, butare not limited to, carcinoma, lymphoma (e.g., Hodgkin's andnon-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. Moreparticular examples of such cancers include squamous cell cancer,small-cell lung cancer, non-small cell lung cancer, adenocarcinoma ofthe lung, squamous carcinoma of the lung, cancer of the peritoneum,hepatocellular cancer, gastrointestinal cancer, appendiceal cancer,pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, livercancer, bladder cancer, hepatoma, breast cancer, colon cancer,colorectal cancer, endometrial or uterine carcinoma, salivary glandcarcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer,thyroid cancer, hepatic carcinoma, leukemia and otherlymphoproliferative disorders, and various types of head and neckcancer.

The term “tumor” as used herein refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all pre-cancerous andcancerous cells and tissues. The terms “cancer,” “cancerous,” “cellproliferative disorder,” “proliferative disorder” and “tumor” are notmutually exclusive as referred to herein.

The term “degenerative muscle disorder” refers to or describes thephysiological condition in muscle-containing animals that is typicallycharacterized by deterioration or weakening of skeletal and/or smoothmuscle such that normal muscular function is reduced. Examples ofdegenerative muscular disorders include, but are not limited to,muscular dystrophy, myotonic dystrophy, myotonia congenita, cachexia,sarcopenia, multiple sclerosis, amyotrophic lateral sclerosis, Isaac'ssyndrome, stiff-person syndrome, familiar periodic paralyses, myopathy,myotonia, rhabdomyolyses, muscle atrophy, and various types of muscleweakness and muscle rigidity.

The term “degenerative nerve disorder” refers to or describes thephysiological condition in nerve-containing animals that is typicallycharacterized by deterioration of nervous tissue or deterioration ofcommunication between cells in nervous tissue. Examples of degenerativenerve disorders include, but are not limited to, neurodegenerativediseases (including, but not limited to, Lewy body disease,postpoliomyelitis syndrome, Shy-Draeger syndrome, olivopontocerebellaratrophy, Parkinson's disease, multiple system atrophy, amyotrophiclateral sclerosis, Guillian-Barre syndrome, Carcot Marie Tooth syndrome,striatonigral degeneration, and nervous cell/tissue destruction causedby or associated with tauopathies, prion diseases, bulbar palsy, motorneuron disease, dementia, and nervous system heterodegenerativedisorders (including, but not limited to, Canavan disease, Huntington'sdisease, neuronal ceroidlipofuscinosis, Alexander's disease, Tourette'ssyndrome, Menkes kinky hair syndrome, Cockayne syndrome,Halervorden-Spatz syndrome, lafora disease, Rett syndrome,hepatolenticular degeneration, Lesch-Nyhan syndrome, andUnverricht-Lundborg syndrome).

In another aspect, the anti-linear linked polyubiquitin antibodies ofthe invention find utility as reagents for detection and isolation oflinear linked polyubiquitin, such as detection of polyubiquitin invarious cell types and tissues, including the determination ofpolyubiquitin density and distribution in cell populations and within agiven cell, and cell sorting based on the presence or amount ofpolyubiquitin. In yet another aspect, the present anti-linear linkedpolyubiquitin antibodies are useful for the development of polyubiquitinantagonists with blocking activity patterns similar to those of thesubject antibodies of the invention. As a further example, anti-linearlinked polyubiquitin antibodies of the invention can be used to identifyother anti-polyubiquitin antibodies that bind substantially the sameantigenic determinant(s) of polyubiquitin as the antibodies exemplifiedherein, including linear and conformational epitopes.

The anti-linear linked polyubiquitin antibodies of the invention can beused in assays based on the physiological pathways in whichpolyubiquitin is involved to screen for small molecule antagonists oflinear linked polyubiquitin function. For example, since linear linkedpolyubiquitin chains are known to be necessary for NFκB activation,(Tokunaga et al., Nature Cell Biol. 11:123-132 (2009)), the activity ofanti-linear linked polyubiquitin antibodies to modulate (up- ordown-regulate) NFκB activation in treated cells or tissues may becompared to the activity of one or more potential small moleculeantagonists of linear linked polyubiquitin in modulating NFκBactivation.

A. Exemplary Anti-Linear Linked Polyubiquitin Antibodies

In one aspect, the invention provides isolated antibodies that bind tolinear or C-terminal to N-terminal-linked polyubiquitin. In certainembodiments, an anti-linear linked polyubiquitin antibody specificallybinds to C-terminal to N-terminal-linked polyubiquitin but does notspecifically bind to monoubiquitin. In certain embodiments, ananti-linear linked polyubiquitin antibody specifically binds toC-terminal to N-terminal-linked polyubiquitin but does not specificallybind to polyubiquitin having a lysine linkage (i.e., K6-, K11-, K27-,K29-, K33-, K48-, and/or K63-linkages).

In one aspect, the invention provides an anti-linear linkedpolyubiquitin antibody comprising an HVR-H1 region comprising thesequence of at least one of SEQ ID NOs: 7, 10, 13, 16, 22 and 73-81. Inone aspect, the invention provides an antibody comprising an HVR-H2region comprising the sequence of at least one of SEQ ID NOs: 8, 11, 14,17, 23, 24 and 82-86. In one aspect, the invention provides an antibodycomprising an HVR-H3 region comprising the sequence of at least one ofSEQ ID NOs: 9, 12, 15, 18 and 87-93.

In one aspect, the invention provides an antibody comprising an HVR-H1region comprising the sequence of at least one of SEQ ID NOs: 7, 10, 13,16, 22 and 73-81, and an HVR-H2 region comprising the sequence of atleast one of SEQ ID NOs: 8, 11, 14, 17, 23, 24 and 82-86. In one aspect,the invention provides an antibody comprising an HVR-H1 regioncomprising the sequence of at least one of SEQ ID NOs: 7, 10, 13, 16, 22and 73-81, and an HVR-H3 region comprising the sequence of at least oneof SEQ ID NOs: 9, 12, 15, 18 and 87-93. In one aspect, the inventionprovides an antibody comprising an HVR-H2 region comprising the sequenceof at least one of SEQ ID NOs: 8, 11, 14, 17, 23, 24 and 81-85 and anHVR-H3 region comprising the sequence of at least one of SEQ ID NOs: 9,12, 15, 18 and 86-92.

In one aspect, the invention provides an antibody comprising an HVR-L1region comprising the sequence of at least one of SEQ ID NOs: 1, 4, 19and 50-57. In one aspect, the invention provides an antibody comprisingan HVR-L2 region comprising the sequence of at least one of SEQ ID NOs:2 and 58-62. In one aspect, the invention provides an antibodycomprising an HVR-L3 region comprising the sequence of at least one ofSEQ ID NOs: 3, 5, 6, 20, 21 and 64-72.

In one aspect, the invention provides an antibody comprising an HVR-L1region comprising the sequence of at least one of SEQ ID NOs: 1, 4, 19and 50-57 and an HVR-L2 region comprising the sequence of at least oneof SEQ ID NOs: 2 and 58-63. In one aspect, the invention provides anantibody comprising an HVR-L1 region comprising the sequence of at leastone of SEQ ID NOs: 1, 4, 19 and 50-57 and an HVR-L3 region comprisingthe sequence of at least one of SEQ ID NOs: 3, 5, 6, 20, 21 and 64-72.In one aspect, the invention provides an antibody comprising an HVR-L2region comprising the sequence of at least one of SEQ ID NOs: 2 and58-63 and an HVR-L3 region comprising the sequence of at least one ofSEQ ID NOs: 3, 5, 6, 20, 21 and 64-72.

In one aspect, the invention provides an antibody comprising at leastone, at least two, at least three, at least four, at least five or allsix of the following:

-   -   (i) an HVR-H1 sequence comprising at least one sequence of SEQ        ID NOs: 7, 10, 13, 16, 22 and 73-81;    -   (ii) an HVR-H2 sequence comprising at least one sequence of SEQ        ID NOs: 8, 11, 14, 17, 23, 24 and 82-86;    -   (iii) an HVR-H3 sequence comprising at least one sequence of SEQ        ID NOs: 9, 12, 15, 18 and 87-93;    -   (iv) an HVR-L1 sequence comprising at least one sequence of SEQ        ID NOs: 1, 4, 19 and 50-57;    -   (v) an HVR-L2 sequence comprising at least one sequence of SEQ        ID NOs: 2 and 58-63; and    -   (vi) an HVR-L3 sequence comprising at least one sequence of SEQ        ID NO: 3, 5, 6, 20, 21 and 64-72.

In one aspect, the invention provides an antibody that specificallybinds linear linked polyubiquitin with high affinity but bindspolyubiquitin some other lysine linkage with substantially reducedaffinity, comprising at least one, at least two, at least three, atleast four, at least five or all six of the following:

-   -   (i) an HVR-H1 sequence comprising at least one sequence of SEQ        ID NOs: 7, 10, 13, 16, 22 and 73-81;    -   (ii) an HVR-H2 sequence comprising at least one sequence of SEQ        ID NOs: 8, 11, 14, 17, 23, 24 and 82-86;    -   (iii) an HVR-H3 sequence comprising at least one sequence of SEQ        ID NOs: 9, 12, 15, 18 and 87-93;    -   (iv) an HVR-L1 sequence comprising at least one sequence of SEQ        ID NOs: 1, 4, 19 and 50-57;    -   (v) an HVR-L2 sequence comprising at least one sequence of SEQ        ID NOs: 2 and 58-63; and    -   (vi) an HVR-L3 sequence comprising at least one sequence of SEQ        ID NO: 3, 5, 6, 20, 21 and 64-72.

In one aspect, the invention provides antibodies comprising heavy chainHVR sequences as depicted in FIG. 2B, 5B, or 9B. In one embodiment, theantibodies comprise light chain HVR sequences as depicted in FIG. 2A,5A, or 9A. In one embodiment, the antibodies comprise heavy chain HVRsequences as depicted in FIG. 2B, 5B, or 9B and light chain HVRsequences as depicted in FIG. 2A, 5A, or 9A. In one embodiment, theantibodies comprise light chain HVR sequences as depicted in Tables 5and 7. In one embodiment, the invention provides antibodies comprisingheavy chain HVR sequences as depicted in Tables 5 and 7. In oneembodiment, the antibodies comprise heavy chain HVR sequences asdepicted in Tables 5 and 7 and light chain HVR sequences as depicted inTables 5 and 7.

Some embodiments of antibodies of the invention comprise a light chainvariable domain of humanized 4D5 antibody (huMAb4D5-8) (HERCEPTIN®,Genentech, Inc., South San Francisco, CA, USA) (also referred to in U.S.Pat. No. 6,407,213 and Lee et al., J. Mol. Biol. (2004), 340(5):1073-93)as depicted in SEQ ID NO: 98 below.

(SEQ ID NO: 98) 1 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu SerAla Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln 

 Val 

 Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr  Ser Ala Ser 

 Leu Tyr Ser Gly Val Pro Ser Arg  Phe Ser Gly Ser 

 Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr  TyrT yr Cys Gln Gln 

 Tyr Thr Thr Pro Pro Thr  Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 107 (HVR residues are underlined)In one embodiment, the huMAb4D5-8 light chain variable domain sequenceis modified at one or more of positions 28, 30, 31, 53, 66, and 91 (Asp,Asn, Thr, Phe, Arg, and His as indicated in bold/italics above,respectively). In one embodiment, the modified huMAb4D5-8 sequencecomprises Ser in position 28, Ser in position 30, Ser in position 31,Ser in position 53, Gly in position 66, and/or Ser in position 91.Accordingly, in one embodiment, an antibody of the invention comprises alight chain variable domain comprising the sequence depicted in SEQ IDNO: 99 below:

(SEQ ID NO: 99) 1 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu SerAla Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln 

 Val 

 Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr  Ser Ala Ser 

 Leu Tyr Ser Gly Val Pro Ser Arg  Phe Ser Gly Ser 

 Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr  Tyr Tyr Cys Gln Gln 

 Tyr Thr Thr Pro Pro Thr  Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 107  (HVR residues are underlined)Substituted residues with respect to huMAb4D5-8 are indicated inbold/italics above.

Antibodies of the invention can comprise any suitable framework variabledomain sequence, provided binding activity to linear linkedpolyubiquitin is substantially retained. For example, in someembodiments, antibodies of the invention comprise a human subgroup IIIheavy chain framework consensus sequence. In one embodiment of theseantibodies, the framework consensus sequence comprises substitution atposition 71, 73, 78 and/or 102. In some embodiments of these antibodies,position 71 is A, 73 is T 78 is A and/or 102 is L. In one embodiment,these antibodies comprise heavy chain variable domain frameworksequences of huMAb4D5-8 (HERCEPTIN®, Genentech, Inc., South SanFrancisco, CA, USA) (also referred to in U.S. Pat. Nos. 6,407,213 &5,821,337, and Lee et al., J. Mol. Biol. (2004), 340(5):1073-93). In oneembodiment, these antibodies further comprise a human κI light chainframework consensus sequence. In one embodiment, these antibodiescomprise at least one, two or all of the light chain HVR sequences ofSEQ ID NOs: 1-6, 19-21 and 50-72. In one embodiment, these antibodiescomprise light chain HVR sequences of huMAb4D5-8 as described in U.S.Pat. Nos. 6,407,213 & 5,821,337.) In one embodiment, these antibodiescomprise light chain variable domain sequences of huMAb4D5-8 (SEQ ID NO:98 and 99) (HERCEPTIN®, Genentech, Inc., South San Francisco, CA, USA)(also referred to in U.S. Pat. Nos. 6,407,213 & 5,821,337, and Lee etal., J. Mol. Biol. (2004), 340(5):1073-93).

In one embodiment, an antibody of the invention is affinity matured toobtain the target binding affinity desired. In one example, an affinitymatured antibody of the invention which specifically binds to linearlinked polyubiquitin with high affinity but binds to polyubiquitinhaving a lysine linkage with substantially reduced affinity comprisessubstitution at HVR-H1 amino acid position 32. In another example, anaffinity matured antibody of the invention which specifically binds tolinear linked polyubiquitin having a lysine linkage with substantiallyreduced affinity comprises substitution at HVR-H2 amino acid positions50, 54 and/or 56. In another example, an affinity matured antibody ofthe invention which specifically binds to linear linked polyubiquitinhaving a lysine linkage with substantially reduced affinity comprisessubstitution at HVR-H3 amino acid position 103. In another example, anaffinity matured antibody of the invention which specifically binds tolinear linked polyubiquitin with high affinity but binds topolyubiquitin having other lysine linkages with substantially reducedaffinity comprises substitution at HVR-L1 amino acid positions 28, 30,31 and/or 32. In another example, an affinity matured antibody of theinvention which specifically binds to linear linked polyubiquitin withhigh affinity but binds to polyubiquitin having other lysine linkageswith substantially reduced affinity comprises substitution at HVR-L3amino acid positions 92, 93 and/or 94. In another example, an affinitymatured antibody of the invention which specifically binds to linearlinked polyubiquitin with high affinity but binds to polyubiquitinhaving other lysine linkages with substantially reduced affinitycomprises substitution at HVR-L2 amino acid position 52.

In one embodiment, an antibody of the invention comprises at least oneheavy chain variable domain sequence of SEQ ID NOs: 29-32, 36-38, 95 and196-198. In one embodiment, an antibody of the invention comprises atleast one light chain variable domain of SEQ ID NOs: 25-28, 33-35, 94and 193-195. In one embodiment, an antibody of the invention comprises aheavy chain variable domain comprising at least one sequence of SEQ IDNOs: 29-32, 36-38, 95 and 196-198 and also comprises a light chainvariable domain comprising at least one sequence of SEQ ID NOs: 25-28,33-35, 94 and 193-195. In other embodiments, an antibody of theinvention corresponding to a particular clone number comprises a heavychain variable domain comprising an HVR-H1, HVR-H2, and HVR-H3 sequenceas set forth in FIG. 2B, 5B or 9B for that clone number. In otherembodiments, an antibody of the invention corresponding to a particularclone number comprises a light chain variable domain comprising anHVR-L1, HVR-L2 and HVR-L3 sequence as set forth in FIGS. 2A, 5A and 9Afor that clone number. In other embodiments, an antibody of theinvention corresponding to a particular clone number comprises a heavychain variable domain comprising an HVR-H1, HVR-H2, and HVR-H3 sequenceas set forth in FIG. 2B, 5B or 9B for that clone number and alsocomprises a light chain variable domain comprising an HVR-L1, HVR-L2 andHVR-L3 sequence as set forth in FIGS. 2A, 5A and 9A for that clonenumber.

In one aspect, the invention provides an antibody that competes with anyof the above-mentioned antibodies for binding to linear linkedpolyubiquitin. In one aspect, the invention provides an antibody thatbinds to the same antigenic determinant on linear linked polyubiquitinas any of the above-mentioned antibodies.

As shown herein, the antibodies of the invention specifically bind to anisolated polyubiquitin having a C-terminal to N-terminal linkage. Asshown herein, the antibodies of the invention also specifically bind topolyubiquitin having a linear C-terminal to N-terminal linkage when thatpolyubiquitin is attached to a heterologous protein.

In any of the above embodiments, an anti-linear linked polyubiquitinantibody is humanized. In one embodiment, an anti-linear linkedpolyubiquitin antibody comprises HVRs as in any of the aboveembodiments, and further comprises an acceptor human framework, e.g. ahuman immunoglobulin framework or a human consensus framework. Inanother embodiment, an anti-linear linked polyubiquitin antibodycomprises HVRs as in any of the above embodiments, and further comprisesa VH comprising an FR1, FR2, FR3, or FR4 sequence of any of SEQ ID NOs:29-32, 36-38 and 95. In another embodiment, an anti-linear linkedpolyubiquitin antibody comprises HVRs as in any of the aboveembodiments, and further comprises a VL comprising an FR1, FR2, FR3, orFR4 sequence of any of SEQ ID NOs: 25-28, 33-35, 94 and 193-195.

In another aspect, an anti-linear linked polyubiquitin antibodycomprises a heavy chain variable domain (VH) sequence having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of any of SEQ ID NOs: 29-32, 26-38,95 and 196-198. In certain embodiments, a VH sequence having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity containssubstitutions (e.g., conservative substitutions), insertions, ordeletions relative to the reference sequence, but an anti-linear linkedpolyubiquitin antibody comprising that sequence retains the ability tobind to linear linked polyubiquitin. In certain embodiments, a total of1 to 10 amino acids have been substituted, inserted and/or deleted inany one of SEQ ID NOs: 29-32, 36-38, 95 and 196-198. In certainembodiments, substitutions, insertions, or deletions occur in regionsoutside the HVRs (i.e., in the FRs). Optionally, the anti-linear linkedpolyubiquitin antibody comprises the VH sequence of any of SEQ ID NOs:29-32, 36-38, 95 and 196-198, including post-translational modificationsof that sequence.

In another aspect, an anti-linear linked polyubiquitin antibody isprovided, wherein the antibody comprises a light chain variable domain(VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100% sequence identity to the amino acid sequence of any of SEQ IDNOs: 25-28, 33-35, 94 and 193-195. In certain embodiments, a VL sequencehaving at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity contains substitutions (e.g., conservative substitutions),insertions, or deletions relative to the reference sequence, but ananti-linear linked polyubiquitin antibody comprising that sequenceretains the ability to bind to linear-linked polyubiquitin. In certainembodiments, a total of 1 to 10 amino acids have been substituted,inserted and/or deleted in any of SEQ ID NOs: 25-28, 33-35, 94 and193-195. In certain embodiments, the substitutions, insertions, ordeletions occur in regions outside the HVRs (i.e., in the FRs).Optionally, the anti-linear linked polyubiquitin antibody comprises theVL sequence in any of SEQ ID NOs: 25-28, 33-35, 94 and 193-195,including post-translational modifications of that sequence.

In another aspect, an anti-linear linked polyubiquitin antibody isprovided, wherein the antibody comprises a VH as in any of theembodiments provided above, and a VL as in any of the embodimentsprovided above. In one embodiment, the antibody comprises the VH and VLsequences in any of SEQ ID NOs: 29-32, 36-38, 95 and 196-198 and SEQ IDNOs: 25-28, 33-35, 94 and 193-195, respectively, includingpost-translational modifications of those sequences.

In a further aspect of the invention, an anti-linear linkedpolyubiquitin antibody according to any of the above embodiments is amonoclonal antibody, including a chimeric, humanized or human antibody.In one embodiment, an anti-linear linked polyubiquitin antibody is anantibody fragment, e.g., a Fv, Fab, Fab′, scFv, diabody, or F(ab′)₂fragment. In another embodiment, the antibody is a full length antibody,e.g., an intact IgG1 antibody or other antibody class or isotype asdefined herein.

Compositions comprising at least one anti-linear linked polyubiquitinantibody or at least one polynucleotide comprising sequences encoding ananti-linear linked polyubiquitin antibody are provided. In certainembodiments, a composition may be a pharmaceutical composition. As usedherein, compositions comprise one or more antibodies that bind to one ormore polyubiquitin and/or one or more polynucleotides comprisingsequences encoding one or more antibodies that bind to one or morepolyubiquitin. These compositions may further comprise suitablecarriers, such as pharmaceutically acceptable excipients includingbuffers, which are well known in the art.

In a further aspect, an anti-linear linked polyubiquitin antibodyaccording to any of the above embodiments may incorporate any of thefeatures, singly or in combination, as described in Sections 1-7 below:

1. Antibody Affinity

In certain embodiments, an antibody provided herein has a dissociationconstant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or≤0.001 nM (e.g. 10⁻⁸ M or less, e.g. from 10⁻⁸ M to 10⁻¹³ M, e.g., from10⁻⁹ M to 10⁻¹³ M).

In one embodiment, Kd is measured by a radiolabeled antigen bindingassay (RIA) performed with the Fab version of an antibody of interestand its antigen as described by the following assay. Solution bindingaffinity of Fabs for antigen is measured by equilibrating Fab with aminimal concentration of (¹²⁵I)-labeled antigen in the presence of atitration series of unlabeled antigen, then capturing bound antigen withan anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol.293:865-881(1999)). To establish conditions for the assay, MICROTITER®multi-well plates (Thermo Scientific) are coated overnight with 5 μg/mlof a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate(pH 9.6), and subsequently blocked with 2% (w/v) bovine serum albumin inPBS for two to five hours at room temperature (approximately 23° C.). Ina non-adsorbent plate (Nunc #269620), 100 pM or 26 pM [¹²⁵I]-antigen aremixed with serial dilutions of a Fab of interest (e.g., consistent withassessment of the anti-VEGF antibody, Fab-12, in Presta et al., CancerRes. 57:4593-4599 (1997)). The Fab of interest is then incubatedovernight; however, the incubation may continue for a longer period(e.g., about 65 hours) to ensure that equilibrium is reached.Thereafter, the mixtures are transferred to the capture plate forincubation at room temperature (e.g., for one hour). The solution isthen removed and the plate washed eight times with 0.1% polysorbate 20(TWEEN-20®) in PBS. When the plates have dried, 150 μl/well ofscintillant (MICROSCINT-20™; Packard) is added, and the plates arecounted on a TOPCOUNT™ gamma counter (Packard) for ten minutes.Concentrations of each Fab that give less than or equal to 20% ofmaximal binding are chosen for use in competitive binding assays.

According to another embodiment, Kd is measured using surface plasmonresonance assays using a BIACORE®-2000 or a BIACORE®-3000 (BIAcore,Inc., Piscataway, NJ) at 25° C. with, e.g., immobilized antigen CM5chips at ˜10 response units (RU). Briefly, carboxymethylated dextranbiosensor chips (CM5, BIACORE, Inc.) are activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 μg/ml (˜0.2μM) before injection at a flow rate of 5 μl/minute to achieveapproximately 10 response units (RU) of coupled protein. Following theinjection of antigen, 1 M ethanolamine is injected to block unreactedgroups. For kinetics measurements, two-fold serial dilutions of Fab(0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20(TWEEN-20™) surfactant (PBST) at 25° C. at a flow rate of approximately25 μl/min. Association rates (k_(on)) and dissociation rates (k_(off))are calculated using a simple one-to-one Langmuir binding model(BIACORE® Evaluation Software version 3.2) by simultaneously fitting theassociation and dissociation sensorgrams. The equilibrium dissociationconstant (Kd) is calculated as the ratio k_(off)/k_(on). See, e.g., Chenet al., J. Mol. Biol. 293:865-881 (1999). If the on-rate exceeds 10⁶ M⁻¹s⁻¹ by the surface plasmon resonance assay above, then the on-rate canbe determined by using a fluorescent quenching technique that measuresthe increase or decrease in fluorescence emission intensity(excitation=295 nm; emission=340 nm, 16 nm band-pass) at 25° C. of a 20nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence ofincreasing concentrations of antigen as measured in a spectrometer, suchas a stop-flow equipped spectrophometer (Aviv Instruments) or a8000-series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) with astirred cuvette. Other coupling chemistries for the target antigen tothe chip surface (e.g., streptavidin/biotin, hydrophobic interaction, ordisulfide chemistry) are also readily available instead of the aminecoupling methodology (CM5 chip) described above, as will be understoodby one of ordinary skill in the art.

2. Antibody Fragments

In certain embodiments, an antibody provided herein is an antibodyfragment. Antibody fragments include, but are not limited to, Fab, Fab′,Fab′-SH, F(ab′)₂, Fv, and scFv fragments, and other fragments describedbelow. For a review of certain antibody fragments, see Hudson et al.Nat. Med 9:129-134 (2003). For a review of scFv fragments, see, e.g.,Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315(1994); see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and5,587,458. For discussion of Fab and F(ab′)2 fragments comprisingsalvage receptor binding epitope residues and having increased in vivohalf-life, see U.S. Pat. No. 5,869,046.

Diabodies are antibody fragments with two antigen-binding sites that maybe bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161;Hudson et al., Nat. Med 9:129-134 (2003); and Hollinger et al., Proc.Natl. Acad Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies arealso described in Hudson et al., Nat. Med 9:129-134 (2003).

Single-domain antibodies are antibody fragments comprising all or aportion of the heavy chain variable domain or all or a portion of thelight chain variable domain of an antibody. In certain embodiments, asingle-domain antibody is a human single-domain antibody (Domantis,Inc., Waltham, MA; see, e.g., U.S. Pat. No. 6,248,516 B1).

Antibody fragments can be made by various techniques, including but notlimited to proteolytic digestion of an intact antibody as well asproduction by recombinant host cells (e.g. E. coli or phage), asdescribed herein.

3. Chimeric and Humanized Antibodies

In certain embodiments, an antibody provided herein is a chimericantibody. Certain chimeric antibodies are described, e.g., in U.S. Pat.No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA,81:6851-6855 (1984)). In one example, a chimeric antibody comprises anon-human variable region (e.g., a variable region derived from a mouse,rat, hamster, rabbit, or non-human primate, such as a monkey) and ahuman constant region. In a further example, a chimeric antibody is a“class switched” antibody in which the class or subclass has beenchanged from that of the parent antibody. Chimeric antibodies includeantigen-binding fragments thereof.

In certain embodiments, a chimeric antibody is a humanized antibody.Typically, a non-human antibody is humanized to reduce immunogenicity tohumans, while retaining the specificity and affinity of the parentalnon-human antibody. Generally, a humanized antibody comprises one ormore variable domains in which HVRs, e.g., CDRs, (or portions thereof)are derived from a non-human antibody, and FRs (or portions thereof) arederived from human antibody sequences. A humanized antibody optionallywill also comprise at least a portion of a human constant region. Insome embodiments, some FR residues in a humanized antibody aresubstituted with corresponding residues from a non-human antibody (e.g.,the antibody from which the HVR residues are derived), e.g., to restoreor improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, e.g., inAlmagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and arefurther described, e.g., in Riechmann et al., Nature 332:323-329 (1988);Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S.Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri etal., Methods 36:25-34 (2005) (describing SDR (a-CDR) grafting); Padlan,Mol. Immunol. 28:489-498 (1991) (describing “resurfacing”); Dall' Acquaet al., Methods 36:43-60 (2005) (describing “FR shuffling”); and Osbournet al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer,83:252-260 (2000) (describing the “guided selection” approach to FRshuffling).

Human framework regions that may be used for humanization include butare not limited to: framework regions selected using the “best-fit”method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); frameworkregions derived from the consensus sequence of human antibodies of aparticular subgroup of light or heavy chain variable regions (see, e.g.,Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta etal. J. Immunol., 151:2623 (1993)); human mature (somatically mutated)framework regions or human germline framework regions (see, e.g.,Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and frameworkregions derived from screening FR libraries (see, e.g., Baca et al., J.Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem.271:22611-22618 (1996)).

4. Human Antibodies

In certain embodiments, an antibody provided herein is a human antibody.Human antibodies can be produced using various techniques known in theart. Human antibodies are described generally in van Dijk and van deWinkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin.Immunol. 20:450-459 (2008).

Human antibodies may be prepared by administering an immunogen to atransgenic animal that has been modified to produce intact humanantibodies or intact antibodies with human variable regions in responseto antigenic challenge. Such animals typically contain all or a portionof the human immunoglobulin loci, which replace the endogenousimmunoglobulin loci, or which are present extrachromosomally orintegrated randomly into the animal's chromosomes. In such transgenicmice, the endogenous immunoglobulin loci have generally beeninactivated. For review of methods for obtaining human antibodies fromtransgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). Seealso, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 describing XENOMOUSE™technology; U.S. Pat. No. 5,770,429 describing HUMAB® technology; U.S.Pat. No. 7,041,870 describing K-M MOUSE® technology, and U.S. PatentApplication Publication No. US 2007/0061900, describing VELOCIMOUSE®technology). Human variable regions from intact antibodies generated bysuch animals may be further modified, e.g., by combining with adifferent human constant region.

Human antibodies can also be made by hybridoma-based methods. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described. (See, e.g., Kozbor J.Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).) Humanantibodies generated via human B-cell hybridoma technology are alsodescribed in Li et al., Proc. Natl. Acad Sci. USA, 103:3557-3562 (2006).Additional methods include those described, for example, in U.S. Pat.No. 7,189,826 (describing production of monoclonal human IgM antibodiesfrom hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268(2006) (describing human-human hybridomas). Human hybridoma technology(Trioma technology) is also described in Vollmers and Brandlein,Histology and Histopathology, 20(3):927-937 (2005) and Vollmers andBrandlein, Methods and Findings in Experimental and ClinicalPharmacology, 27(3):185-91 (2005).

Human antibodies may also be generated by isolating Fv clone variabledomain sequences selected from human-derived phage display libraries.Such variable domain sequences may then be combined with a desired humanconstant domain. Techniques for selecting human antibodies from antibodylibraries are described below.

5. Library-Derived Antibodies

Antibodies of the invention may be isolated by screening combinatoriallibraries for antibodies with the desired activity or activities. Forexample, a variety of methods are known in the art for generating phagedisplay libraries and screening such libraries for antibodies possessingthe desired binding characteristics. Such methods are reviewed, e.g., inHoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien etal., ed., Human Press, Totowa, N J, 2001) and further described, e.g.,in the McCafferty et al., Nature 348:552-554; Clackson et al., Nature352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992);Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo,ed., Human Press, Totowa, N J, 2003); Sidhu et al., J. Mol. Biol.338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093(2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472(2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132(2004).

In certain phage display methods, repertoires of VH and VL genes areseparately cloned by polymerase chain reaction (PCR) and recombinedrandomly in phage libraries, which can then be screened forantigen-binding phage as described in Winter et al., Ann. Rev. Immunol.,12: 433-455 (1994). Phage typically display antibody fragments, eitheras single-chain Fv (scFv) fragments or as Fab fragments. Libraries fromimmunized sources provide high-affinity antibodies to the immunogenwithout the requirement of constructing hybridomas. Alternatively, thenaive repertoire can be cloned (e.g., from human) to provide a singlesource of antibodies to a wide range of non-self and also self antigenswithout any immunization as described by Griffiths et al., EMBO J, 12:725-734 (1993). Finally, naive libraries can also be made syntheticallyby cloning unrearranged V-gene segments from stem cells, and using PCRprimers containing random sequence to encode the highly variable CDR3regions and to accomplish rearrangement in vitro, as described byHoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patentpublications describing human antibody phage libraries include, forexample: U.S. Pat. No. 5,750,373, and US Patent Publication Nos.2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598,2007/0237764, 2007/0292936, and 2009/0002360.

Antibodies or antibody fragments isolated from human antibody librariesare considered human antibodies or human antibody fragments herein.

6. Multispecific Antibodies

In certain embodiments, an antibody provided herein is a multispecificantibody, e.g. a bispecific antibody. Multispecific antibodies aremonoclonal antibodies that have binding specificities for at least twodifferent sites. In certain embodiments, one of the bindingspecificities is for linear linked polyubiquitin and the other is forany other antigen. In certain embodiments, bispecific antibodies maybind to two different epitopes of linear linked polyubiquitin.Bispecific antibodies may also be used to localize cytotoxic agents tocells which express linear linked polyubiquitin. Bispecific antibodiescan be prepared as full length antibodies or antibody fragments.

Techniques for making multispecific antibodies include, but are notlimited to, recombinant co-expression of two immunoglobulin heavychain-light chain pairs having different specificities (see Milstein andCuello, Nature 305: 537 (1983)), WO 93/08829, and Traunecker et al.,EMBO J. 10: 3655 (1991)), and “knob-in-hole” engineering (see, e.g.,U.S. Pat. No. 5,731,168). Multi-specific antibodies may also be made byengineering electrostatic steering effects for making antibodyFc-heterodimeric molecules (WO 2009/089004A1); cross-linking two or moreantibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennanet al., Science, 229: 81 (1985)); using leucine zippers to producebi-specific antibodies (see, e.g., Kostelny et al., 20 J. Immunol.,148(5):1547-1553 (1992)); using “diabody” technology for makingbispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv)dimers (see, e.g. Gruber et al., J. Immunol., 152:5368 (1994)); andpreparing trispecific antibodies as described, e.g., in Tutt et al. J.Immunol. 147: 60 (1991).

Engineered antibodies with three or more functional antigen bindingsites, including “Octopus antibodies,” are also included herein (see,e.g. US 2006/0025576A1).

The antibody or fragment herein also includes a “Dual Acting FAb” or“DAF” comprising an antigen binding site that binds to linear linkedpolyubiquitin as well as another, different antigen (see, US2008/0069820, for example).

7. Antibody Variants

In certain embodiments, amino acid sequence variants of the antibodiesprovided herein are contemplated. For example, it may be desirable toimprove the binding affinity and/or other biological properties of theantibody. Amino acid sequence variants of an antibody may be prepared byintroducing appropriate modifications into the nucleotide sequenceencoding the antibody, or by peptide synthesis. Such modificationsinclude, for example, deletions from, and/or insertions into and/orsubstitutions of residues within the amino acid sequences of theantibody. Any combination of deletion, insertion, and substitution canbe made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics, e.g., antigen-binding.

a) Substitution, Insertion, and Deletion Variants

In certain embodiments, antibody variants having one or more amino acidsubstitutions are provided. Sites of interest for substitutionalmutagenesis include the HVRs and FRs. Conservative substitutions areshown in Table 1 under the heading of “conservative substitutions.” Moresubstantial changes are provided in Table 1 under the heading of“exemplary substitutions,” and as further described below in referenceto amino acid side chain classes. Amino acid substitutions may beintroduced into an antibody of interest and the products screened for adesired activity, e.g., retained/improved antigen binding, decreasedimmunogenicity, or improved ADCC or CDC.

TABLE 1 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine;Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine LeuAmino acids may be grouped according to common side-chain properties:

-   -   (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;    -   (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;    -   (3) acidic: Asp, Glu;    -   (4) basic: His, Lys, Arg;    -   (5) residues that influence chain orientation: Gly, Pro;    -   (6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g. a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther study will have modifications (e.g., improvements) in certainbiological properties (e.g., increased affinity, reduced immunogenicity)relative to the parent antibody and/or will have substantially retainedcertain biological properties of the parent antibody. An exemplarysubstitutional variant is an affinity matured antibody, which may beconveniently generated, e.g., using phage display-based affinitymaturation techniques such as those described herein. Briefly, one ormore HVR residues are mutated and the variant antibodies displayed onphage and screened for a particular biological activity (e.g. bindingaffinity).

Alterations (e.g., substitutions) may be made in HVRs, e.g., to improveantibody affinity. Such alterations may be made in HVR “hotspots,” i.e.,residues encoded by codons that undergo mutation at high frequencyduring the somatic maturation process (see, e.g., Chowdhury, MethodsMol. Biol. 207:179-196 (2008)), and/or SDRs (a-CDRs), with the resultingvariant VH or VL being tested for binding affinity. Affinity maturationby constructing and reselecting from secondary libraries has beendescribed, e.g., in Hoogenboom et al. in Methods in Molecular Biology178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001).) In someembodiments of affinity maturation, diversity is introduced into thevariable genes chosen for maturation by any of a variety of methods(e.g., error-prone PCR, chain shuffling, or oligonucleotide-directedmutagenesis). A secondary library is then created. The library is thenscreened to identify any antibody variants with the desired affinity.Another method to introduce diversity involves HVR-directed approaches,in which several HVR residues (e.g., 4-6 residues at a time) arerandomized. HVR residues involved in antigen binding may be specificallyidentified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3and CDR-L3 in particular are often targeted.

In certain embodiments, substitutions, insertions, or deletions mayoccur within one or more HVRs so long as such alterations do notsubstantially reduce the ability of the antibody to bind antigen. Forexample, conservative alterations (e.g., conservative substitutions asprovided herein) that do not substantially reduce binding affinity maybe made in HVRs. Such alterations may be outside of HVR “hotspots” orSDRs. In certain embodiments of the variant VH and VL sequences providedabove, each HVR either is unaltered, or contains no more than one, twoor three amino acid substitutions.

A useful method for identification of residues or regions of an antibodythat may be targeted for mutagenesis is called “alanine scanningmutagenesis” as described by Cunningham and Wells (1989) Science,244:1081-1085. In this method, a residue or group of target residues(e.g., charged residues such as arg, asp, his, lys, and glu) areidentified and replaced by a neutral or negatively charged amino acid(e.g., alanine or polyalanine) to determine whether the interaction ofthe antibody with antigen is affected. Further substitutions may beintroduced at the amino acid locations demonstrating functionalsensitivity to the initial substitutions. Alternatively, oradditionally, a crystal structure of an antigen-antibody complex toidentify contact points between the antibody and antigen. Such contactresidues and neighboring residues may be targeted or eliminated ascandidates for substitution. Variants may be screened to determinewhether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue. Other insertionalvariants of the antibody molecule include the fusion to the N- orC-terminus of the antibody to an enzyme (e.g. for ADEPT) or apolypeptide which increases the serum half-life of the antibody.

b) Glycosylation Variants

In certain embodiments, an antibody provided herein is altered toincrease or decrease the extent to which the antibody is glycosylated.Addition or deletion of glycosylation sites to an antibody may beconveniently accomplished by altering the amino acid sequence such thatone or more glycosylation sites is created or removed.

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. Native antibodies produced by mammalian cellstypically comprise a branched, biantennary oligosaccharide that isgenerally attached by an N-linkage to Asn297 of the CH2 domain of the Fcregion. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). Theoligosaccharide may include various carbohydrates, e.g., mannose,N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as afucose attached to a GlcNAc in the “stem” of the biantennaryoligosaccharide structure. In some embodiments, modifications of theoligosaccharide in an antibody of the invention may be made in order tocreate antibody variants with certain improved properties.

In one embodiment, antibody variants are provided having a carbohydratestructure that lacks fucose attached (directly or indirectly) to an Fcregion. For example, the amount of fucose in such antibody may be from1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amountof fucose is determined by calculating the average amount of fucosewithin the sugar chain at Asn297, relative to the sum of allglycostructures attached to Asn 297 (e. g. complex, hybrid and highmannose structures) as measured by MALDI-TOF mass spectrometry, asdescribed in WO 2008/077546, for example. Asn297 refers to theasparagine residue located at about position 297 in the Fc region (Eunumbering of Fc region residues); however, Asn297 may also be locatedabout +3 amino acids upstream or downstream of position 297, i.e.,between positions 294 and 300, due to minor sequence variations inantibodies. Such fucosylation variants may have improved ADCC function.See, e.g., US Patent Publication Nos. US 2003/0157108 (Presta, L.); US2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publicationsrelated to “defucosylated” or “fucose-deficient” antibody variantsinclude: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614;US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki etal. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech.Bioeng. 87: 614 (2004). Examples of cell lines capable of producingdefucosylated antibodies include Lec13 CHO cells deficient in proteinfucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986);US Pat Appl No US 2003/0157108 A1, Presta, L; and WO 2004/056312 A1,Adams et al., especially at Example 11), and knockout cell lines, suchas alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see,e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. etal., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003/085107).

Antibodies variants are further provided with bisected oligosaccharides,e.g., in which a biantennary oligosaccharide attached to the Fc regionof the antibody is bisected by GlcNAc. Such antibody variants may havereduced fucosylation and/or improved ADCC function. Examples of suchantibody variants are described, e.g., in WO 2003/011878 (Jean-Mairet etal.); U.S. Pat. No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umanaet al.). Antibody variants with at least one galactose residue in theoligosaccharide attached to the Fc region are also provided. Suchantibody variants may have improved CDC function. Such antibody variantsare described, e.g., in WO 1997/30087 (Patel et al.); WO 1998/58964(Raju, S.); and WO 1999/22764 (Raju, S.).

c) Fc Region Variants

In certain embodiments, one or more amino acid modifications may beintroduced into the Fc region of an antibody provided herein, therebygenerating an Fc region variant. The Fc region variant may comprise ahuman Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fcregion) comprising an amino acid modification (e.g. a substitution) atone or more amino acid positions.

In certain embodiments, the invention contemplates an antibody variantthat possesses some but not all effector functions, which make it adesirable candidate for applications in which the half life of theantibody in vivo is important yet certain effector functions (such ascomplement and ADCC) are unnecessary or deleterious. In vitro and/or invivo cytotoxicity assays can be conducted to confirm thereduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theantibody lacks FcγR binding (hence likely lacking ADCC activity), butretains FcRn binding ability. The primary cells for mediating ADCC, NKcells, express Fc(RIII only, whereas monocytes express Fc(RI, Fc(RII andFc(RIII. FcR expression on hematopoietic cells is summarized in Table 3on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991).Non-limiting examples of in vitro assays to assess ADCC activity of amolecule of interest is described in U.S. Pat. No. 5,500,362 (see, e.g.Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) andHellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985);5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361(1987)). Alternatively, non-radioactive assays methods may be employed(see, for example, ACTI™ non-radioactive cytotoxicity assay for flowcytometry (CellTechnology, Inc. Mountain View, CA; and CytoTox 96®non-radioactive cytotoxicity assay (Promega, Madison, WI). Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and Natural Killer (NK) cells. Alternatively, oradditionally, ADCC activity of the molecule of interest may be assessedin vivo, e.g., in a animal model such as that disclosed in Clynes et al.Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays mayalso be carried out to confirm that the antibody is unable to bind C1qand hence lacks CDC activity. See, e.g., C1q and C3c binding ELISA in WO2006/029879 and WO 2005/100402. To assess complement activation, a CDCassay may be performed (see, for example, Gazzano-Santoro et al., J.Immunol. Methods 202:163 (1996); Cragg, M. S. et al., Blood101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie, Blood103:2738-2743 (2004)). FcRn binding and in vivo clearance/half lifedeterminations can also be performed using methods known in the art(see, e.g., Petkova, S. B. et al., Int'l. Immunol. 18(12):1759-1769(2006)).

Antibodies with reduced effector function include those withsubstitution of one or more of Fc region residues 238, 265, 269, 270,297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fcmutants with substitutions at two or more of amino acid positions 265,269, 270, 297 and 327, including the so-called “DANA” Fc mutant withsubstitution of residues 265 and 297 to alanine (U.S. Pat. No.7,332,581).

Certain antibody variants with improved or diminished binding to FcRsare described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, andShields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).)

In certain embodiments, an antibody variant comprises an Fc region withone or more amino acid substitutions which improve ADCC, e.g.,substitutions at positions 298, 333, and/or 334 of the Fc region (EUnumbering of residues).

In some embodiments, alterations are made in the Fc region that resultin altered (i.e., either improved or diminished) C1q binding and/orComplement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat.No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

Antibodies with increased half lives and improved binding to theneonatal Fc receptor (FcRn), which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) andKim et al., J. Immunol. 24:249 (1994)), are described inUS2005/0014934A1 (Hinton et al.). Those antibodies comprise an Fc regionwith one or more substitutions therein which improve binding of the Fcregion to FcRn. Such Fc variants include those with substitutions at oneor more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307,311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434,e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826).

See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat. Nos.5,648,260; 5,624,821; and WO 94/29351 concerning other examples of Fcregion variants.

d) Cysteine Engineered Antibody Variants

In certain embodiments, it may be desirable to create cysteineengineered antibodies, e.g., “thioMAbs,” in which one or more residuesof an antibody are substituted with cysteine residues. In particularembodiments, the substituted residues occur at accessible sites of theantibody. By substituting those residues with cysteine, reactive thiolgroups are thereby positioned at accessible sites of the antibody andmay be used to conjugate the antibody to other moieties, such as drugmoieties or linker-drug moieties, to create an immunoconjugate, asdescribed further herein. In certain embodiments, any one or more of thefollowing residues may be substituted with cysteine: V205 (Kabatnumbering) of the light chain; A118 (EU numbering) of the heavy chain;and S400 (EU numbering) of the heavy chain Fc region. Cysteineengineered antibodies may be generated as described, e.g., in U.S. Pat.No. 7,521,541.

e) Antibody Derivatives

In certain embodiments, an antibody provided herein may be furthermodified to contain additional nonproteinaceous moieties that are knownin the art and readily available. The moieties suitable forderivatization of the antibody include but are not limited to watersoluble polymers. Non-limiting examples of water soluble polymersinclude, but are not limited to, polyethylene glycol (PEG), copolymersof ethylene glycol/propylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.Polyethylene glycol propionaldehyde may have advantages in manufacturingdue to its stability in water. The polymer may be of any molecularweight, and may be branched or unbranched. The number of polymersattached to the antibody may vary, and if more than one polymer isattached, they can be the same or different molecules. In general, thenumber and/or type of polymers used for derivatization can be determinedbased on considerations including, but not limited to, the particularproperties or functions of the antibody to be improved, whether theantibody derivative will be used in a therapy under defined conditions,etc.

In another embodiment, conjugates of an antibody and nonproteinaceousmoiety that may be selectively heated by exposure to radiation areprovided. In one embodiment, the nonproteinaceous moiety is a carbonnanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605(2005)). The radiation may be of any wavelength, and includes, but isnot limited to, wavelengths that do not harm ordinary cells, but whichheat the nonproteinaceous moiety to a temperature at which cellsproximal to the antibody-nonproteinaceous moiety are killed.

B. Recombinant Methods and Compositions

Antibodies may be produced using recombinant methods and compositions,e.g., as described in U.S. Pat. No. 4,816,567. In one embodiment,isolated nucleic acid encoding an anti-linear linked polyubiquitinantibody described herein is provided. Such nucleic acid may encode anamino acid sequence comprising the VL and/or an amino acid sequencecomprising the VH of the antibody (e.g., the light and/or heavy chainsof the antibody). In a further embodiment, one or more vectors (e.g.,expression vectors) comprising such nucleic acid are provided. In afurther embodiment, a host cell comprising such nucleic acid isprovided. In one such embodiment, a host cell comprises (e.g., has beentransformed with): (1) a vector comprising a nucleic acid that encodesan amino acid sequence comprising the VL of the antibody and an aminoacid sequence comprising the VH of the antibody, or (2) a first vectorcomprising a nucleic acid that encodes an amino acid sequence comprisingthe VL of the antibody and a second vector comprising a nucleic acidthat encodes an amino acid sequence comprising the VH of the antibody.In one embodiment, the host cell is eukaryotic, e.g. a Chinese HamsterOvary (CHO) cell or lymphoid cell (e.g., Y0, NS0, Sp20 cell). In oneembodiment, a method of making an anti-linear linked polyubiquitinantibody is provided, wherein the method comprises culturing a host cellcomprising a nucleic acid encoding the antibody, as provided above,under conditions suitable for expression of the antibody, and optionallyrecovering the antibody from the host cell (or host cell culturemedium).

For recombinant production of an anti-linear linked polyubiquitinantibody, nucleic acid encoding an antibody, e.g., as described above,is isolated and inserted into one or more vectors for further cloningand/or expression in a host cell. Such nucleic acid may be readilyisolated and sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of the antibody).

Suitable host cells for cloning or expression of antibody-encodingvectors include prokaryotic or eukaryotic cells described herein. Forexample, antibodies may be produced in bacteria, in particular whenglycosylation and Fc effector function are not needed. For expression ofantibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat.Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods inMolecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, describing expression of antibody fragments in E.coli.) After expression, the antibody may be isolated from the bacterialcell paste in a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors, including fungi and yeast strains whoseglycosylation pathways have been “humanized,” resulting in theproduction of an antibody with a partially or fully human glycosylationpattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li etal., Nat. Biotech. 24:210-215 (2006).

Suitable host cells for the expression of glycosylated antibody are alsoderived from multicellular organisms (invertebrates and vertebrates).Examples of invertebrate cells include plant and insect cells. Numerousbaculoviral strains have been identified which may be used inconjunction with insect cells, particularly for transfection ofSpodoptera frugiperda cells.

Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Pat.Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429(describing PLANTIBODIES™ technology for producing antibodies intransgenic plants).

Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977));baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells asdescribed, e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); monkeykidney cells (CV1); African green monkey kidney cells (VERO-76); humancervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo ratliver cells (BRL 3A); human lung cells (W138); human liver cells (HepG2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., inMather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; andFS4 cells. Other useful mammalian host cell lines include Chinesehamster ovary (CHO) cells, including DHFR⁻ CHO cells (Urlaub et al.,Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines suchas Y0, NS0 and Sp2/0. For a review of certain mammalian host cell linessuitable for antibody production, see, e.g., Yazaki and Wu, Methods inMolecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa,NJ), pp. 255-268 (2003).

C. Assays

Anti-linear linked polyubiquitin antibodies provided herein may beidentified, screened for, or characterized for their physical/chemicalproperties and/or biological activities by various assays known in theart.

1. Binding Assays and Other Assays

In one aspect, an antibody of the invention is tested for its antigenbinding activity, e.g., by known methods such as ELISA, Western blot,etc.

In another aspect, competition assays may be used to identify anantibody that competes with, e.g., any of Fabs or antibodies describedherein such as 1E3, 1D8, 1F4, 1A10, 1D8.3C2, 1D8.3F8. 1D8.4F5, 1F11,2A2, 2C11, 2H5, 3E4, 4C9, 4E4, 4G7, 3F5, 3A7 and 4C10 or hybridantibodies as described herein, for example 4E4/T110A, 4C9/T110A,4G7/Y102G, 1F11/3F5, 1F11/3E4. 1F11/4G7, 2C11/3E4, 2C11/3F5, 2C11/4G7,2H5/3E4, 2H5/3F5, 2H5/4G7, 1F11/3F5/Y102L for binding to linear linkedpolyubiquitin. In certain embodiments, such a competing antibody bindsto the same epitope (e.g., a linear or a conformational epitope) that isbound by any of Fabs or antibodies described herein such as 1E3, 1D8,1F4, 1A10, 1D8.3C2, 1D8.3F8. 1D8.4F5, 1F11, 2A2, 2C11, 2H5, 3E4, 4C9,4E4, 4G7, 3F5, 3A7 and 4C10 or hybrid antibodies as described herein,for example 4E4/T110A, 4C9/T110A, 4G7/Y102G, 1F11/3F5, 1F11/3E4.1F11/4G7, 2C11/3E4, 2C11/3F5, 2C11/4G7, 2H5/3E4, 2H5/3F5, 2H5/4G7,1F11/3F5/Y102L for binding to linear linked polyubiquitin. Detailedexemplary methods for mapping an epitope to which an antibody binds areprovided in Morris (1996) “Epitope Mapping Protocols,” in Methods inMolecular Biology vol. 66 (Humana Press, Totowa, NJ).

In an exemplary competition assay, immobilized linear linkedpolyubiquitin is incubated in a solution comprising a first labeledantibody that binds to linear linked polyubiquitin (e.g., antibodies1E3, 1D8, 1F4, 1A10, 1D8.3C2, 1D8.3F8. 1D8.4F5, 1F11, 2A2, 2C11, 2H5,3E4, 4C9, 4E4, 4G7, 3F5, 3A7 and 4C10 or hybrid antibodies 4E4/T110A,4C9/T110A, 4G7/Y102G, 1F11/3F5, 1F11/3E4. 1F11/4G7, 2C11/3E4, 2C11/3F5,2C11/4G7, 2H5/3E4, 2H5/3F5, 2H5/4G7, 1F11/3F5/Y102L for binding tolinear linked polyubiquitin and a second unlabeled antibody that isbeing tested for its ability to compete with the first antibody forbinding to linear linked polyubiquitin. The second antibody may bepresent in a hybridoma supernatant. As a control, immobilized linearlinked polyubiquitin is incubated in a solution comprising the firstlabeled antibody but not the second unlabeled antibody. After incubationunder conditions permissive for binding of the first antibody to linearlinked polyubiquitin, excess unbound antibody is removed, and the amountof label associated with immobilized linear linked polyubiquitin ismeasured. If the amount of label associated with immobilized linearlinked polyubiquitin is substantially reduced in the test samplerelative to the control sample, then that indicates that the secondantibody is competing with the first antibody for binding to linearlinked polyubiquitin. See Harlow and Lane (1988) Antibodies: ALaboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold SpringHarbor, NY).

2. Activity Assays

In one aspect, assays are provided for identifying anti-linear linkedpolyubiquitin antibodies thereof having biological activity. Biologicalactivity may include, e.g., modulating the rate of degradation of linearlinked polyubiquitinated proteins in a cell or tissue, and modulatingthe rate of cell cycle progression of a cell. Antibodies having suchbiological activity in vivo and/or in vitro are also provided.

In certain embodiments, an antibody of the invention is tested for suchbiological activity.

D. Immunoconjugates

The invention also provides immunoconjugates comprising an anti-linearlinked polyubiquitin antibody herein conjugated to one or more cytotoxicagents, such as chemotherapeutic agents or drugs, growth inhibitoryagents, toxins (e.g., protein toxins, enzymatically active toxins ofbacterial, fungal, plant, or animal origin, or fragments thereof), orradioactive isotopes.

In one embodiment, an immunoconjugate is an antibody-drug conjugate(ADC) in which an antibody is conjugated to one or more drugs, includingbut not limited to a maytansinoid (see U.S. Pat. Nos. 5,208,020,5,416,064 and European Patent EP 0 425 235 B1); an auristatin such asmonomethylauristatin drug moieties DE and DF (MMAE and MMAF) (see U.S.Pat. Nos. 5,635,483 and 5,780,588, and 7,498,298); a dolastatin; acalicheamicin or derivative thereof (see U.S. Pat. Nos. 5,712,374,5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode etal., Cancer Res. 58:2925-2928 (1998)); an anthracycline such asdaunomycin or doxorubicin (see Kratz et al., Current Med Chem.13:477-523 (2006); Jeffrey et al., Bioorganic & Med Chem. Letters16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagyet al., Proc. Natl. Acad Sci. USA 97:829-834 (2000); Dubowchik et al.,Bioorg. & Med Chem. Letters 12:1529-1532 (2002); King et al., J. MedChem. 45:4336-4343 (2002); and U.S. Pat. No. 6,630,579); methotrexate;vindesine; a taxane such as docetaxel, paclitaxel, larotaxel, tesetaxel,and ortataxel; a trichothecene; and CC1065.

In another embodiment, an immunoconjugate comprises an antibody asdescribed herein conjugated to an enzymatically active toxin or fragmentthereof, including but not limited to diphtheria A chain, nonbindingactive fragments of diphtheria toxin, exotoxin A chain (from Pseudomonasaeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), Momordica charantiainhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.

In another embodiment, an immunoconjugate comprises an antibody asdescribed herein conjugated to a radioactive atom to form aradioconjugate. A variety of radioactive isotopes are available for theproduction of radioconjugates. Examples include At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰,Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu.When the radioconjugate is used for detection, it may comprise aradioactive atom for scintigraphic studies, for example tc99m or I123,or a spin label for nuclear magnetic resonance (NMR) imaging (also knownas magnetic resonance imaging, mri), such as iodine-123 again,iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17,gadolinium, manganese or iron.

Conjugates of an antibody and cytotoxic agent may be made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238:1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026. Thelinker may be a “cleavable linker” facilitating release of a cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari et al., Cancer Res. 52:127-131(1992); U.S. Pat. No. 5,208,020) may be used.

The immunuoconjugates or ADCs herein expressly contemplate, but are notlimited to such conjugates prepared with cross-linker reagentsincluding, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS,MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS,sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB(succinimidyl-(4-vinylsulfone)benzoate) which are commercially available(e.g., from Pierce Biotechnology, Inc., Rockford, IL., U.S.A).

E. Methods and Compositions for Diagnostics and Detection

In certain embodiments, any of the anti-linear linked polyubiquitinantibodies provided herein is useful for detecting the presence oflinear linked polyubiquitin in a biological sample. The term “detecting”as used herein encompasses quantitative or qualitative detection. Incertain embodiments, a biological sample comprises a cell or tissue,such as, but not limited to, a tumor cell, a muscle cell or a nervecell.

In one embodiment, an anti-linear linked polyubiquitin antibody for usein a method of diagnosis or detection is provided. In a further aspect,a method of detecting the presence of linear linked polyubiquitin in abiological sample is provided. In certain embodiments, the methodcomprises contacting the biological sample with an anti-linear linkedpolyubiquitin antibody as described herein under conditions permissivefor binding of the anti-linear linked polyubiquitin antibody to apolyubiquitin or polyubiquitinated protein, and detecting whether acomplex is formed between the anti-linear linked polyubiquitin antibodyand the polyubiquitin or polyubiquitinated protein. Such method may bean in vitro or in vivo method. In one embodiment, an anti-linear linkedpolyubiquitin antibody is used to select subjects eligible for therapywith an anti-linear linked polyubiquitin antibody, e.g. where linearlinked polyubiquitin is a biomarker for selection of patients.

Exemplary disorders that may be diagnosed using an antibody of theinvention include cell-cycle-related diseases or disorders, which may bea disease or disorder associated with aberrantly increased cell cycleprogression or a disease or disorder associated with aberrantlydecreased cell cycle progression. In one aspect, a disease or disorderassociated with aberrantly increased cell cycle progression is cancer.In another aspect, a disease or disorder associated with aberrantlydecreased cell cycle progression is, e.g., a degenerative muscledisorder or a degenerative nerve disorder.

In certain embodiments, labeled anti-linear linked polyubiquitinantibodies are provided. Labels include, but are not limited to, labelsor moieties that are detected directly (such as fluorescent,chromophoric, electron-dense, chemiluminescent, and radioactive labels),as well as moieties, such as enzymes or ligands, that are detectedindirectly, e.g., through an enzymatic reaction or molecularinteraction. Exemplary labels include, but are not limited to, theradioisotopes ³²P, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I, fluorophores such as rareearth chelates or fluorescein and its derivatives, rhodamine and itsderivatives, dansyl, umbelliferone, luceriferases, e.g., fireflyluciferase and bacterial luciferase (U.S. Pat. No. 4,737,456),luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP),alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme,saccharide oxidases, e.g., glucose oxidase, galactose oxidase, andglucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricaseand xanthine oxidase, coupled with an enzyme that employs hydrogenperoxide to oxidize a dye precursor such as HRP, lactoperoxidase, ormicroperoxidase, biotin/avidin, spin labels, bacteriophage labels,stable free radicals, and the like.

F. Pharmaceutical Formulations

Pharmaceutical formulations of an anti-linear linked polyubiquitinantibody as described herein are prepared by mixing such antibody havingthe desired degree of purity with one or more optional pharmaceuticallyacceptable carriers (Remington's Pharmaceutical Sciences 16th edition,Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueoussolutions. Pharmaceutically acceptable carriers are generally nontoxicto recipients at the dosages and concentrations employed, and include,but are not limited to: buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride; benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as polyethylene glycol(PEG). Exemplary pharmaceutically acceptable carriers herein furtherinclude interstitial drug dispersion agents such as solubleneutral-active hyaluronidase glycoproteins (sHASEGP), for example, humansoluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®,Baxter International, Inc.). Certain exemplary sHASEGPs and methods ofuse, including rHuPH20, are described in US Patent Publication Nos.2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined withone or more additional glycosaminoglycanases such as chondroitinases.

Exemplary lyophilized antibody formulations are described in U.S. Pat.No. 6,267,958. Aqueous antibody formulations include those described inU.S. Pat. No. 6,171,586 and WO2006/044908, the latter formulationsincluding a histidine-acetate buffer.

The formulation herein may also contain more than one active ingredientsas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother. For example, it may be desirable to further provide one or morechemotherapeutic agents. Such active ingredients are suitably present incombination in amounts that are effective for the purpose intended.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules.

The formulations to be used for in vivo administration are generallysterile. Sterility may be readily accomplished, e.g., by filtrationthrough sterile filtration membranes.

G. Therapeutic Methods and Compositions

Any of the anti-linear linked polyubiquitin antibodies provided hereinmay be used in therapeutic methods.

In one aspect, an anti-linear linked polyubiquitin antibody for use as amedicament is provided. In further aspects, an anti-linear linkedpolyubiquitin antibody for use in treating disorders associated withaberrant cell cycle regulation (including, but not limited to,proliferation disorders such as cancer and hypotrophy disordersincluding, but not limited to, degenerative muscle disorders anddegenerative nerve disorders) is provided. In certain embodiments, ananti-linear linked polyubiquitin antibody for use in a method oftreatment is provided. In certain embodiments, the invention provides ananti-linear linked polyubiquitin antibody for use in a method oftreating an individual having a disorder associated with aberrant cellcycle regulation, comprising administering to the individual aneffective amount of the anti-linear linked polyubiquitin antibody. Inone such embodiment, the method further comprises administering to theindividual an effective amount of at least one additional therapeuticagent, e.g., as described below. In further embodiments, the inventionprovides an anti-linear linked polyubiquitin antibody for use inmodulating cell cycle regulation such that the rate of cell cycleprogression is adjusted. In certain embodiments, the invention providesan anti-linear linked polyubiquitin antibody for use in a method ofmodulating the rate of cell cycle progression in an individualcomprising administering to the individual an effective of theanti-linear linked polyubiquitin antibody to modulate cell cycleprogression and thereby adjust the rate of cell division. An“individual” according to any of the above embodiments is preferably ahuman.

In a further aspect, the invention provides for the use of ananti-linear linked polyubiquitin antibody in the manufacture orpreparation of a medicament. In one embodiment, the medicament is fortreatment of disorders associated with aberrant cell cycle regulation(including, but not limited to, proliferation disorders such as cancerand hypotrophy disorders including, but not limited to, degenerativemuscle disorders and degenerative nerve disorders). In a furtherembodiment, the medicament is for use in a method of treating a disorderassociated with aberrant cell cycle regulation comprising administeringto an individual having such a disorder an effective amount of themedicament. In one such embodiment, the method further comprisesadministering to the individual an effective amount of at least oneadditional therapeutic agent, e.g., as described below. In a furtherembodiment, the medicament is for modulating the rate of cell cycleprogression. In a further embodiment, the medicament is for use in amethod of modulating the rate of cell cycle progression in an individualcomprising administering to the individual an amount effective of themedicament to adjust the rate of cellular division. An “individual”according to any of the above embodiments may be a human.

In a further aspect, the invention provides a method for treating adisorder associated with aberrant cell cycle regulation. In oneembodiment, the method comprises administering to an individual havingsuch a disorder associated with aberrant cell cycle regulation aneffective amount of an anti-linear linked polyubiquitin antibody. In onesuch embodiment, the method further comprises administering to theindividual an effective amount of at least one additional therapeuticagent, as described below. An “individual” according to any of the aboveembodiments may be a human.

In a further aspect, the invention provides pharmaceutical formulationscomprising any of the anti-linear linked polyubiquitin antibodiesprovided herein, e.g., for use in any of the above therapeutic methods.In one embodiment, a pharmaceutical formulation comprises any of theanti-linear linked polyubiquitin antibodies provided herein and apharmaceutically acceptable carrier. In another embodiment, apharmaceutical formulation comprises any of the anti-linear linkedpolyubiquitin antibodies provided herein and at least one additionaltherapeutic agent, e.g., as described below.

Antibodies of the invention can be used either alone or in combinationwith other agents in a therapy. For instance, an antibody of theinvention may be co-administered with at least one additionaltherapeutic agent. In certain embodiments, an additional therapeuticagent is a chemotherapeutic agent.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines suchas benzodopa, carboquone, meturedopa, and uredopa; ethylenimines andmethylamelamines including altretamine, triethylenemelamine,trietylenephosphoramide, triethiylenethiophosphoramide andtrimethylolomelamine; acetogenins (especially bullatacin andbullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®);beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin(including the synthetic analogue topotecan (HYCAMTIN®), CPT-11(irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including itsadozelesin, carzelesin and bizelesin synthetic analogues);podophyllotoxin; podophyllinic acid; teniposide; cryptophycins(particularly cryptophycin 1 and cryptophycin 8); dolastatin;duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1);eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;antibiotics such as the enediyne antibiotics (e. g., calicheamicin,especially calicheamicin gamma1I and calicheamicin omegaIl (see, e.g.,Agnew, Chem Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, includingdynemicin A; an esperamicin; as well as neocarzinostatin chromophore andrelated chromoprotein enediyne antiobiotic chromophores),aclacinomysins, actinomycin, authramycin, azaserine, bleomycins,cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis,dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS NaturalProducts, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium;tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine;trichothecenes (especially T-2 toxin, verracurin A, roridin A andanguidine); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); thiotepa; taxoids, e.g., TAXOL® paclitaxel(Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™Cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Illinois), andTAXOTERE® doxetaxel (Rhône-Poulenc Rorer, Antony, France); chloranbucil;gemcitabine (GEMZAR®); 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine(VELBAN®); platinum; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine (ONCOVIN®); oxaliplatin; leucovovin; vinorelbine(NAVELBINE®); novantrone; edatrexate; daunomycin; aminopterin;ibandronate; topoisomerase inhibitor RFS 2000; difluorometlhylornithine(DMFO); retinoids such as retinoic acid; capecitabine (XELODA®);pharmaceutically acceptable salts, acids or derivatives of any of theabove; as well as combinations of two or more of the above such as CHOP,an abbreviation for a combined therapy of cyclophosphamide, doxorubicin,vincristine, and prednisolone, and FOLFOX, an abbreviation for atreatment regimen with oxaliplatin (ELOXATIN™) combined with 5-FU andleucovovin.

Also included in this definition are anti-hormonal agents that act toregulate, reduce, block, or inhibit the effects of hormones that canpromote the growth of cancer, and are often in the form of systemic, orwhole-body treatment. They may be hormones themselves. Examples includeanti-estrogens and selective estrogen receptor modulators (SERMs),including, for example, tamoxifen (including NOLVADEX® tamoxifen),EVISTA® raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene,keoxifene, LY117018, onapristone, and FARESTON® toremifene;anti-progesterones; estrogen receptor down-regulators (ERDs); agentsthat function to suppress or shut down the ovaries, for example,leutinizing hormone-releasing hormone (LHRH) agonists such as LUPRON®and ELIGARD® leuprolide acetate, goserelin acetate, buserelin acetateand tripterelin; other anti-androgens such as flutamide, nilutamide andbicalutamide; and aromatase inhibitors that inhibit the enzymearomatase, which regulates estrogen production in the adrenal glands,such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE®megestrol acetate, AROMASIN® exemestane, formestanie, fadrozole,RIVISOR® vorozole, FEMARA® letrozole, and ARIMIDEX® anastrozole. Inaddition, such definition of chemotherapeutic agents includesbisphosphonates such as clodronate (for example, BONEFOS® or OSTAC®),DIDROCAL® etidronate, NE-58095, ZOMETA® zoledronic acid/zoledronate,FOSAMAX® alendronate, AREDIA® pamidronate, SKELID® tiludronate, orACTONEL® risedronate; as well as troxacitabine (a 1,3-dioxolanenucleoside cytosine analog); antisense oligonucleotides, particularlythose that inhibit expression of genes in signaling pathways implicatedin abherant cell proliferation, such as, for example, PKC-alpha, Raf,H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such asTHERATOPE® vaccine and gene therapy vaccines, for example, ALLOVECTIN®vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; LURTOTECAN®topoisomerase 1 inhibitor; ABARELIX® rmRH; lapatinib ditosylate (anErbB-2 and EGFR dual tyrosine kinase small-molecule inhibitor also knownas GW572016); and pharmaceutically acceptable salts, acids orderivatives of any of the above.

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate formulations), and separate administration, in which case,administration of the antibody of the invention can occur prior to,simultaneously, and/or following, administration of the additionaltherapeutic agent and/or adjuvant. Antibodies of the invention can alsobe used in combination with radiation therapy.

An antibody of the invention (and any additional therapeutic agent) canbe administered by any suitable means, including parenteral,intrapulmonary, and intranasal, and, if desired for local treatment,intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. Dosing can be by any suitable route, e.g.by injections, such as intravenous or subcutaneous injections, dependingin part on whether the administration is brief or chronic. Variousdosing schedules including but not limited to single or multipleadministrations over various time-points, bolus administration, andpulse infusion are contemplated herein.

Antibodies of the invention would be formulated, dosed, and administeredin a fashion consistent with good medical practice. Factors forconsideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. Theantibody need not be, but is optionally formulated with one or moreagents currently used to prevent or treat the disorder in question. Theeffective amount of such other agents depends on the amount of antibodypresent in the formulation, the type of disorder or treatment, and otherfactors discussed above. These are generally used in the same dosagesand with administration routes as described herein, or about from 1 to99% of the dosages described herein, or in any dosage and by any routethat is empirically/clinically determined to be appropriate.

The location of the binding target of an antibody of the invention maybe taken into consideration in preparation and administration of theantibody. When the binding target is an intracellular molecule, certainembodiments of the invention provide for the antibody or antigen-bindingfragment thereof to be introduced into the cell where the binding targetis located. In one embodiment, an antibody of the invention can beexpressed intracellularly as an intrabody. The term “intrabody,” as usedherein, refers to an antibody or antigen-binding portion thereof that isexpressed intracellularly and that is capable of selectively binding toa target molecule, as described in Marasco, Gene Therapy 4: 11-15(1997); Kontermann, Methods 34: 163-170 (2004); U.S. Pat. Nos. 6,004,940and 6,329,173; U.S. Patent Application Publication No. 2003/0104402, andPCT Publication No. WO2003/077945. Intracellular expression of anintrabody is effected by introducing a nucleic acid encoding the desiredantibody or antigen-binding portion thereof (lacking the wild-typeleader sequence and secretory signals normally associated with the geneencoding that antibody of antigen-binding fragment) into a target cell.Any standard method of introducing nucleic acids into a cell may beused, including, but not limited to, microinjection, ballisticinjection, electroporation, calcium phosphate precipitation, liposomes,and transfection with retroviral, adenoviral, adeno-associated viral andvaccinia vectors carrying the nucleic acid of interest. One or morenucleic acids encoding all or a portion of an anti-polyubiquitinantibody of the invention can be delivered to a target cell, such thatone or more intrabodies are expressed which are capable of intracellularbinding to a polyubiquitin and modulation of one or morepolyubiquitin-mediated cellular pathways.

In another embodiment, internalizing antibodies are provided. Antibodiescan possess certain characteristics that enhance delivery of antibodiesinto cells, or can be modified to possess such characteristics.Techniques for achieving this are known in the art. For example,cationization of an antibody is known to facilitate its uptake intocells (see, e.g., U.S. Pat. No. 6,703,019). Lipofections or liposomescan also be used to deliver the antibody into cells. Where antibodyfragments are used, the smallest inhibitory fragment that specificallybinds to the binding domain of the target protein is generallyadvantageous. For example, based upon the variable-region sequences ofan antibody, peptide molecules can be designed that retain the abilityto bind the target protein sequence. Such peptides can be synthesizedchemically and/or produced by recombinant DNA technology. See, e.g.,Marasco et al., Proc. Natl. Acad. Sci. USA 90: 7889-7893 (1993).

Entry of modulator polypeptides into target cells can be enhanced bymethods known in the art. For example, certain sequences, such as thosederived from HIV Tat or the Antennapedia homeodomain protein are able todirect efficient uptake of heterologous proteins across cell membranes.See, e.g., Chen et al., Proc. Natl. Acad. Sci. USA 96: 4325-4329 (1999).

When the binding target is located in the brain, certain embodiments ofthe invention provide for the antibody or antigen-binding fragmentthereof to traverse the blood-brain barrier. Certain neurodegenerativediseases are associated with an increase in permeability of theblood-brain barrier, such that the antibody or antigen-binding fragmentcan be readily introduced to the brain. When the blood-brain barrierremains intact, several art-known approaches exist for transportingmolecules across it, including, but not limited to, physical methods,lipid-based methods, and receptor and channel-based methods.

Physical methods of transporting the antibody or antigen-bindingfragment across the blood-brain barrier include, but are not limited to,circumventing the blood-brain barrier entirely, or by creating openingsin the blood-brain barrier. Circumvention methods include, but are notlimited to, direct injection into the brain (see, e.g., Papanastassiouet al., Gene Therapy 9: 398-406 (2002); interstitialinfusion/convection-enhanced delivery (see, e.g., Bobo et al., Proc.Natl. Acad. Sci. USA 91: 2076-2080 (1994)), and implanting a deliverydevice in the brain (see, e.g., Gill et al., Nature Med. 9: 589-595(2003); and Gliadel Wafers™, Guildford Pharmaceutical). Methods ofcreating openings in the barrier include, but are not limited to,ultrasound (see, e.g., U.S. Patent Publication No. 2002/0038086),osmotic pressure (e.g., by administration of hypertonic mannitol(Neuwelt, E. A., Implication of the Blood-Brain Barrier and itsManipulation, vols. 1 & 2, Plenum Press, N.Y. (1989))), permeabilizationby, e.g., bradykinin or permeabilizer A-7 (see, e.g., U.S. Pat. Nos.5,112,596, 5,268,164, 5,506,206, and 5,686,416), and transfection ofneurons that straddle the blood-brain barrier with vectors containinggenes encoding the antibody or antigen-binding fragment (see, e.g., U.S.Patent Publication No. 2003/0083299).

Lipid-based methods of transporting the antibody or antigen-bindingfragment across the blood-brain barrier include, but are not limited to,encapsulating the antibody or antigen-binding fragment in liposomes thatare coupled to antibody binding fragments that bind to receptors on thevascular endothelium of the blood-brain barrier (see, e.g., U.S. PatentApplication Publication No. 20020025313), and coating the antibody orantigen-binding fragment in low-density lipoprotein particles (see,e.g., U.S. Patent Application Publication No. 20040204354) orapolipoprotein E (see, e.g., U.S. Patent Application Publication No.20040131692).

Receptor and channel-based methods of transporting the antibody orantigen-binding fragment across the blood-brain barrier include, but arenot limited to, using glucocorticoid blockers to increase permeabilityof the blood-brain barrier (see, e.g., U.S. Patent ApplicationPublication Nos. 2002/0065259, 2003/0162695, and 2005/0124533);activating potassium channels (see, e.g., U.S. Patent ApplicationPublication No. 2005/0089473); inhibiting ABC drug transporters (see,e.g., U.S. Patent Application Publication No. 2003/0073713); coatingantibodies with a transferrin and modulating activity of the one or moretransferrin receptors (see, e.g., U.S. Patent Application PublicationNo. 2003/0129186), and cationizing the antibodies (see, e.g., U.S. Pat.No. 5,004,697).

For the prevention or treatment of disease, the appropriate dosage of anantibody of the invention (when used alone or in combination with one ormore other additional therapeutic agents) will depend on the type ofdisease to be treated, the type of antibody, the severity and course ofthe disease, whether the antibody is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the antibody, and the discretion of the attendingphysician. The antibody is suitably administered to the patient at onetime or over a series of treatments. Depending on the type and severityof the disease, about 1 μg/kg to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg) ofantibody can be an initial candidate dosage for administration to thepatient, whether, for example, by one or more separate administrations,or by continuous infusion. One typical daily dosage might range fromabout 1 μg/kg to 100 mg/kg or more, depending on the factors mentionedabove. For repeated administrations over several days or longer,depending on the condition, the treatment would generally be sustaineduntil a desired suppression of disease symptoms occurs. One exemplarydosage of the antibody would be in the range from about 0.05 mg/kg toabout 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg,4.0 mg/kg or 10 mg/kg (or any combination thereof) may be administeredto the patient. Such doses may be administered intermittently, e.g.every week or every three weeks (e.g. such that the patient receivesfrom about two to about twenty, or e.g. about six doses of theantibody). An initial higher loading dose, followed by one or more lowerdoses may be administered. However, other dosage regimens may be useful.The progress of this therapy is easily monitored by conventionaltechniques and assays.

It is understood that any of the above formulations or therapeuticmethods may be carried out using an immunoconjugate of the invention inplace of or in addition to an anti-linear linked polyubiquitin antibody.

H. Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, IV solution bags, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition which is by itself or combined with anothercomposition effective for treating, preventing and/or diagnosing thecondition and may have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is an antibody of the invention. The label or package insertindicates that the composition is used for treating the condition ofchoice. Moreover, the article of manufacture may comprise (a) a firstcontainer with a composition contained therein, wherein the compositioncomprises an antibody of the invention; and (b) a second container witha composition contained therein, wherein the composition comprises afurther cytotoxic or otherwise therapeutic agent. The article ofmanufacture in this embodiment of the invention may further comprise apackage insert indicating that the compositions can be used to treat aparticular condition. Alternatively, or additionally, the article ofmanufacture may further comprise a second (or third) containercomprising a pharmaceutically-acceptable buffer, such as bacteriostaticwater for injection (BWFI), phosphate-buffered saline, Ringer's solutionand dextrose solution. It may further include other materials desirablefrom a commercial and user standpoint, including other buffers,diluents, filters, needles, and syringes.

It is understood that any of the above articles of manufacture mayinclude an immunoconjugate of the invention in place of or in additionto an anti-linear linked polyubiquitin antibody.

III. Examples

The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, giventhe general description provided above.

Example 1: Isolation and Characterization of Anti-Linear PolyubiquitinAntibodies

A) Antigen Generation

The antigen used for sorting the phage display libraries was lineardiubiquitin (Boston Biochem). Linear diubiquitin is a head to tailfusion of two ubiquitins through a peptide bond between the carboxyterminus of the first ubiquitin and the amino terminus of the secondubiquitin.

B) Naïve Library Sorting

The naïve YSGX Fab phage display library was subjected to four rounds ofsorting against linear diubiquitin. No enrichment was observed afterfour rounds of sorting (see Table 2). The YSGX Fab phage display librarycontains randomized amino acids in all three heavy chain CDRs and lightchain CDR L3 (see U.S. Published Patent Application No. 2005-0106667 andFellouse F. et al. JMB 373:924-40 (2007)), and is based on a humanizedantibody 4D5.

The naïve common light chain YSGX Fab phage display library wassubjected to four rounds of sorting against linear diubiquitin.Twenty-five-fold enrichment was observed after four rounds of sorting(see Table 2). The common light chain YSGX Fab phage display librarycontains randomized amino acids in all three heavy chain CDRs asdescribed for the YSGX library (see U.S. Published Patent ApplicationNo. 2005-0106667 and Fellouse F. et al. JMB 373:924-40 (2007)), howeverthe light chain sequence is fixed as a modified version of humanizedantibody 4D5.

The naïve VH Fab phage display library was subjected to four rounds ofsorting against linear diubiquitin. Eight hundred-fold enrichment wasobserved after four rounds of sorting (see Table 1). The VH Fab phagedisplay library contains randomized amino acids in all three heavy chainCDRs (see U.S. Published Patent Application No. 2005/0119455 and Lee C.W. et al. JMB 340:1073-93 (2004)), and is based on a humanized antibody4D5.

Linear diubiquitin (Boston Biochem) was immobilized on a 96-wellMaxisorb immunoplate (NUNC). Plates were coated overnight at 4° C. with5 μg/mL linear diubiquitin in 50 mM sodium carbonate buffer, pH 9.6. Thecoated plates were blocked with 200 μL/well of 2.5% milk in phosphatebuffered saline (PBS) containing 0.05% Tween 20 (PBST) for one hour at25° C. with shaking. The naïve phage libraries were precipitated fromglycerol stocks with 1/5 volume of 20% polyethylene glycol (PEG)/2.5MNaCl, resuspended in 2.5% milk/PBST, and incubated at 25° C. for onehour while the plate was blocking. After one hour, the blocking bufferwas dumped off of the plate and 100 μL/well of phage in 2.5% milk/PBSTwas added and incubated at 25° C. for four hours with shaking. Afterbinding, the plate was washed ten times with PBST by manually fillingthe wells and dumping off the buffer between washes. Phage were elutedwith 150 μL/well of 50 mM HCl/500 mM KCl for 30 minutes at 25° C. withshaking. The elution was neutralized with 150 μL/well of 1 M Tris, pH7.5 and subsequently propagated in XL1-Blue (Agilent) Escherichia coli(E. coli) with the addition of M13K07 helper phage.

Amplified phage were used for additional rounds of selection againstlinear diubiquitin as described above. In rounds two through four,soluble ubiquitin of different forms were added to the phage forcounterselection. In the second round, 10 μg/mL of soluble monoubiquitin(Boston Biochem) was used. In the third and fourth rounds, 10 μg/mL eachof soluble monoubiquitin (Boston Biochem), K11-linked diubiquitin(Genentech), K48-linked polyubiquitin 2-7 (Boston Biochem), andK63-linked polyubiquitin 2-7 chains (Boston Biochem) were used.Enrichment was calculated for rounds two through four by comparing thenumber of phage recovered with linear diubiquitin compared to anuncoated well. Enrichment was observed in rounds two through four forthe common light chain library and the VH library, but not the YSGXlibrary (see Table 2).

TABLE 2 Library Round 2 Round 3 Round 4 Common light 2X  2X  25X chainYSGX YSGX 0X  0X  0X VH 3X 120X 800X

Ninety-six individual clones from the second round of sorting of the VHlibrary, 192 clones from the third round of sorting of the VH library,96 clones from the fourth round of sorting of the VH library, and 192clones from the fourth round of sorting of the common light chainlibrary were screened. Since no enrichment was seen for the YSGXlibrary, no clones were screened from this library. Individual cloneswere grown up in a 96-well format in 1 mL of 2YT broth containing 50μg/mL carbenicillin and 1×10¹⁰ phage/mL M13K07 helper phage at 37° C.overnight with shaking. Cells were pelleted by spinning at 3000 rpm forten minutes. Supernatants from those cultures were used inhigh-throughput phage spot enzyme linked immunosorbant assays (ELISAs)for binding to linear diubiquitin (Boston Biochem), monoubiquitin(Boston Biochem), K11-linked diubiquitin (Genentech), K48-linkedpolyubiquitin 2-7 (Boston Biochem), K63-linked polyubiquitin 2-7 (BostonBiochem), an anti-gD antibody (Genentech), or an uncoated well. All ofthe Fab libraries contain a carboxy-terminal gD tag on the light chainwhich allows for assessment of display level by anti-gD antibodybinding. The panel of ubiquitin proteins was immobilized on 384-wellMaxisorb immunoplates (NUNC). Plates were coated at 4° C. overnight with2 μg/mL of each protein in 50 mM sodium carbonate buffer, pH 9.6. Thecoated plates were blocked with 60 μL/well of 2.5% milk in PBST for onehour at 25° C. with shaking. After one hour, the blocking buffer wasdumped off of the plate and 20 μL/well of PBST and 10 μL/well of phagesupernatant were added. Plates were incubated at 25° C. for one hourwith shaking. The plate was then washed six times with PBST by manuallyfilling the wells and dumping off the wash buffer. A 1:5,000 dilution ofan anti-M13 horseradish peroxidase (HRP)-conjugated secondary antibody(GE Healthcare) in PBST was used for detection of phage binding. 30μL/well of the secondary dilution was added and the plate was incubatedat 25° C. for 30 minutes with shaking. The plate was then washed sixtimes with PBST and twice with PBS, both manually. Bound secondaryantibody was detected using a TMB substrate (KPL) followed by quenchingwith an equal volume of 1 M phosphoric acid. The absorbance was read at450 nm.

From the common light chain library, several weak lineardiubiquitin-specific binders were identified (see FIG. 1 ). The lightand heavy chain variable domains of these clones were sequenced. Twounique sequences (1F4 (SEQ ID NOs:27 and 31) and 1A10 (SEQ ID NOs:28 and31)) were identified, however, based on the light chain sequence it wasdetermined that one of these clones (1F4) was actually from the YSGXlibrary that does not contain a fixed light chain (see FIG. 2A). Fromthe VH library several clones showing strong linear diubiquitin bindingwith weaker binding to K63-linked polyubiquitin were identified (seeFIG. 1 ). The heavy chain variable domains of these VH library cloneswere sequenced. The CDR H1, CDR H2, and CDR H3 sequences are expected tobe clone-specific, whereas, the heavy chain framework sequences shouldbe identical, based on the VH library design. The entire light chainsequence (both framework and CDRs) is expected to be invariant due tothe library design. The CDR L1 sequence is RASQDVSTAVA (SEQ ID NO:1),the CDR L2 sequence is SASFLYS (SEQ ID NO:2), and the CDR L3 sequence isQQSYTTPPT (SEQ ID NO:3). Two unique heavy chain sequences wereidentified (1D8 (SEQ ID NO:30) and 1E3 (SEQ ID NO:29)) (see FIG. 2B).

C) Conversion of the Phagemids to Monovalent Fab Display

The 1D8 and 1E3 phagemid clones from the VH library were converted frombivalent Fab-zip format to monovalent Fab display for affinitymaturation purposes. The leucine zipper between the end of the CH1constant domain and the start of gene III (gpIII) was removed usingKunkel mutagenesis (see Kunkel, Proc. Natl. Acad. Sci. USA 82:488(1985)). Mutagenic oligonucleotide F220-delzip(TCTTGTGACAAAACTCACAGTGGCGGTGGCTCTGGT) (SEQ ID NO:100) was combined with1 μg of 1D8 or 1E3 phagemid Kunkel DNA.

The 1A10 and 1F4 clones from the common light chain library and the YSGXlibrary, respectively, were converted from bivalent Fab-C format tomonovalent Fab display for affinity maturation purposes. The cysteinebetween the end of the CH1 constant domain and gpIII was removed usingKunkel mutagenesis. Mutagenic oligonucleotide F1120-delCGRP(TGTGACAAAACTCACCTCAGTGGCGGTGGCTCTGGTTCCGGTGATTTTGATTATGAA AAG) (SEQ IDNO:101) was combined with 1 μg of 1A10 or 1F4 phagemid Kunkel DNA. Theresulting monovalent Fab phagemids were used to produce phage for IC₅₀ELISAs.

D) Phage IC₅₀ ELISAs

Phage displaying monovalent Fab for 1D8, 1E3, 1A10, and 1F4 were testedin an IC₅₀ ELISA to get an estimate of relative affinity for lineardiubiquitin. An initial titer ELISA was done to determine the amount ofphage at which a signal of OD₄₅₀=0.5 would be achieved. Lineardiubiquitin (Boston Biochem) was immobilized on 96-well Maxisorbimmunoplates (NUNC). Linear diubiquitin was coated overnight at 4° C. at1 μg/mL in 50 mM sodium carbonate buffer, pH 9.6. The coated plates wereblocked with 200 μL/well of 2.5% milk in PBST for one hour at 25° C.with shaking. Twelve two-fold serial dilutions of the phage were made in2.5% milk in PBST from OD₂₆₈=4.0 to OD₂₆₈=0.002. After one hour, theblocking buffer was dumped off of the plate and 100 μL/well of eachphage dilution was added and incubated at 25° C. for 15 minutes withshaking. The plate was then washed six times with PBST using a platewasher. A 1:5000 dilution of an anti-M13 phage-HRP-conjugated secondaryantibody (GE Healthcare) in PBST was used for detection of phagebinding. 100 μL/well of the secondary dilution was added and the platewas incubated at 25° C. for 30 minutes with shaking. The plate was thenwashed 12 times with PBST using a plate washer and twice with PBSmanually. Bound secondary antibody was detected using a TMB substrate(KPL) followed by quenching with an equal volume of 1 M phosphoric acid.The absorbance was read at 450 nm. The concentration of phage at whichan OD₄₅₀=0.5 was OD₂₆₈=1.0 of phage for clone 1F4, an OD₂₆₈=0.125 forclone 1D8, and an OD₂₆₈=0.5 for clone 1E3. For clone 1A10, even at phageOD₂₆₈=4.0 the OD₄₅₀ was only 0.376. This binding is quite weak andtherefore clone 1A10 was not pursued further. Two-fold serial dilutionsof soluble linear diubiquitin from 10 μM to 5 nM for clone 1F4 andthree-fold serial dilutions of soluble linear diubiquitin from 10 μM to56 μM for clones 1D8 and 1E3 plus the selected phage concentrations in2.5% milk in PBST were incubated at 25° C. for one hour with shaking.The amount of unbound phage at each linear diubiquitin concentration wasthen measured by incubating the mixtures with a 96-well Maxisorbimmunoplate that had been coated with 1 μg/mL linear diubiquitin andblocked with 2.5% milk in PBST. The phage/linear diubiquitin mixture wasincubated on the plate for 15 minutes at 25° C. with shaking. The platewas then washed six times with PBST using a plate washer. A 1:5000dilution of an anti-M13 phage-HRP-conjugated secondary antibody (GEHealthcare) in PBST was used for detection of phage binding. 100 μL/wellof the secondary dilution was added and the plate was incubated at 25°C. for 30 minutes with shaking. The plate was then washed 12 times withPBST using a plate washer and twice with PBS manually. Bound secondaryantibody was detected using a TMB substrate (KPL) followed by quenchingwith an equal volume of 1 M phosphoric acid. The absorbance was read at450 nm. The absorbance was plotted against soluble linear diubiquitinconcentration and shows that for 1F4 the IC₅₀ is greater than 10 μM (seeFIG. 3 ), for 1D8 the IC₅₀ is near 5 μM (see FIG. 3 ), and for 1E3 theIC₅₀ is 80 nM (see FIG. 3 ).

E) Fab Production

Clones derived from the Fab phage display libraries are expressed underthe control of the E. coli alkaline phosphatase (PhoA) promoter. Boththe light chain and the heavy chain contain an amino-terminal bacterialstII signal sequence to allow secretion in E. coli and are expressedfrom a single phagemid vector. The heavy chain carboxyl terminus isfused in-frame to the C-terminus of gene product III (gpIII) of the M13bacteriophage, allowing for display of a monovalent Fab fragment onphage. In order to express soluble Fab, a stop codon was introduced intothe 1D8 and 1E3 monovalent phagemids between the end of the CH1 constantdomain of the Fab and the start of gpIII. Mutagenic oligonucleotides:5′-FabdelzipTAA (CCCAAATCTTGTGACAAAACTCACACATAAAGTGGCGGTGGCTCTGGTTCCGGTG) (SEQ ID NO:102) and 3′-FabdelzipTAA(CACCGGAACCAGAGCCACCGCCACTTTATGTGTGAGTT TTGTCACAAGATTTGGG) (SEQ IDNO:103) were used to insert the stop codon using the QuikChange®Lightning Site-Directed Mutagenesis kit (Agilent). The resulting solubleFab expression plasmids were transformed into the E. coli strain 62A7(Genentech) and plated on solid agar containing carbenicillin. Singlecolonies were used to inoculate 25 mL of 2YT broth containing 50 μg/mLcarbenicillin. The culture was grown overnight at 37° C. and 5 mL wereused to inoculate 500 mL of complete C.R.A.P. media (3.57 g (NH4)2SO4,0.71 g sodium citrate 2H2O, 1.07 g KCl, 5.36 g yeast extract(certified), 5.36 g Hycase SF (Sheffield), pH adjusted to 7.3 byaddition of KOH and volume adjusted to 872 mL with ultrapure water,autoclaved, cooled to 55° C., to which was added (per L) 110 mL 1M MOPSpH 7.3, 11 mL 50% glucose, and 7 mL 1M MgSO4) with 50 μg/mLcarbenicillin. The cultures were grown at 30° C. for 24 hours withshaking. Cells were harvested by centrifugation and pellets were storedat −20° C. The Fab was purified by resuspending the cell pellet in 35 mLof cold wash buffer (Phosphate Buffered Saline (PBS)+150 mM NaCl)containing 10 μg/mL DNaseI (Invitrogen), 0.2 mg/mL lysozyme (USB), and 1mM phenylmethylsulphonylfluoride (PMSF) (Calbiochem). The pellet wasresuspended by vortexing rapidly. To allow complete lysis the cells wereincubated for 15 minutes at 25° C. Cell debris was pelleted bycentrifugation and lysate was loaded on 1 mL protein A-sepharose (GEHealthcare) column preequilibrated with cold wash buffer. The column waswashed with 50 mL of cold wash buffer, eluted with 3 mL of 0.1 M aceticacid, and neutralized with 150 μL of 1 M Tris, pH 11.0. The Fab wasconcentrated using Amicon Ultra-15 centrifugal filter units (10 kDacut-off, Millipore). The resulting Fab concentration was determinedspectrophotometrically (1 OD₂₈₀=1.5 mg/mL).

F) Fab Western Blot

The 1D8 and 1E3 Fabs (from Example 1E) were tested for binding to lineardiubiquitin (Boston Biochem), monoubiquitin (Boston Biochem), K11-linked diubiquitin (Genentech), K48-linked diubiquitin (BostonBiochem), and K63-linked diubiquitin (Boston Biochem) in a western blot.1 μg of each protein in 1×LDS buffer (Invitrogen) with reducing agentwas heated at 70° C. for ten minutes and run on 4-12% NuPAGE Bis Tris1.0 mm gels in MES buffer (Invitrogen) in duplicate. Gels weretransferred at 30 V constant for 1.5 hours by wet transfer in 10%methanol and 1×NuPAGE transfer buffer (Invitrogen) to 0.2 μmnitrocellulose (Invitrogen). Non-specific binding sites on the membraneswere blocked by incubation in 5% milk in PBST for 1.5 hours at 25° C.with shaking. The membranes were then incubated in 5 μg/mL of 1D8 or 1E3Fab in 5% milk in PBST for one hour at 25° C. with shaking. The membranewas washed three times in PBST with shaking. The Fabs were detected byincubating the membrane in a 1:10000 dilution of a goat anti-human Fabfragment-specific HRP-conjugated secondary antibody (Sigma Aldrich) in5% milk in PBST for one hour at 25° C. with shaking. The membranes werethen washed three times in PBST followed by one wash in PBS. Thesecondary antibody was detected using Super Signal West Picochemiluminescent substrate (Thermo Scientific) followed by exposure ofthe blots to film. The 1E3 Fab detects only the linear diubiquitin butnot monoubiquitin, K11-linked diubiquitin, K48-linked diubiquitin, orK63-linked diubiquitin (see FIG. 4 ). The 1D8 Fab did not detect anyforms of ubiquitin by western blot (see FIG. 4 ).

G) Affinity Analysis of Isolated 1D8 and 1E3 Fabs

The affinity of the 1D8 and 1E3 Fabs (from Example 1E) was analyzed bysurface plasmon resonance (SPR) using a BIACORE™ 3000 (GE Healthcare).Approximately 120 resonance units (RUs) of linear diubiquitin (BostonBiochem), K48-linked diubiquitin (Boston Biochem), and K63-linkeddiubiquitin (Boston Biochem) were immobilized on flow cell two, flowcell three, and flow cell four, respectively, of a CM5 chip using theamine coupling protocol supplied by the manufacturer. Flow cell one wasactivated and ethanolamine blocked without immobilizing protein, to beused for reference subtraction. Two-fold serial dilutions (0.5-500 nM)of 1E3 Fab in 10 mM Hepes, pH 7.2, 150 mM NaCl, and 0.01% Tween 20(HBST) were injected (60 μL total at a flow rate of 30 μL/minute) overeach flow cell using HBST as the running buffer. The signal for eachflow cell was recorded and the reference signal was subtracted.Different regeneration conditions were scouted. Even with 10 mM HCl thechip surface could not be completely regenerated back to baseline. When10 mM glycine, pH 1.7 was tested this altered the chip surface anddecreased the binding capacity.

An alternative approach was tested using a Fab capture method on aBIACORE™ 3000 (GE Healthcare). Approximately 11,000 resonance units(RUs) of an anti-human Fab capture antibody (GE Healthcare) wereimmobilized on flow cells one and two of a CM5 chip using the aminecoupling protocol supplied by the manufacturer. 10 μL of 10 μg/mL Fab in10 mM Hepes, pH 7.2, 150 mM NaCl, and 0.01% Tween 20 (HBST) was injectedat a flow rate of 10 μL/minute over flow cell two, resulting in captureof approximately 430 RUs of Fab. Flow cell one had only the captureantibody on it to serve as a reference subtraction. Two-fold serialdilutions (1-1000 nM) of linear diubiquitin (Boston Biochem) orK63-linked diubiquitin (Boston Biochem) in HBST were injected (60 μLtotal at a flow rate of 30 μL/minute) over flow cells one and two. Thesignal for each flow cell was recorded and the reference signal wassubtracted. Following a dissociation period of four minutes, the chipsurface was regenerated with two injections of 30 μL of 10 mM glycine,pH 2.1 at a flow rate of 30 μL/minute. Data were difficult to fit to anybinding model because the diubiquitin did not fully dissociate from thechip. In addition the association rates were very fast and binding didnot reach a plateau even at the highest concentration of diubiquitinused. Therefore it is difficult to estimate a K_(D) for these Fabs.

Example 2—Affinity Maturation of 1F4, 1D8, and 1E3

A) Stop Template Generation

A TAA stop codon was inserted separately into either CDR L1, CDR L2, CDRL3, CDR H3, or both CDRs L3 and H3 (resulting in L1, L2, L3, H3, andL3/H3 stop templates, respectively) for library synthesis using Kunkelmutagenesis. Stop codons force diversity within a particular CDR loop byrequiring repair of the stop in order to get full length Fab expressionand display on phage. The stop codon mutagenic oligonucleotides listedbelow were combined with 1 μg of the corresponding monovalent phagemidKunkel DNA. The resulting monovalent Fab phagemid stop templates wereused for affinity maturation library generation.

For clones 1D8 and 1E3 the CDR L1 stop mutagenic oligonucleotide used toinsert a TAA stop codon at position 24 (Kabat numbering) within CDR L1of the light chain was 4D5LC1.stop (GTCACCATCACCTGCTAAGCCAGTCAGGATGTG)(SEQ ID NO:104). For clones 1D8 and 1E3 the CDR L2 stop mutagenicoligonucleotide used to insert a TAA stop codon at position 50 (Kabatnumbering) within CDR L2 of the light chain was 4D5LC2.stop(GAAGCTTCTGATTTACTAAGCATCCTTCCTCTAC) (SEQ ID NO:105). For clones 1D8 and1E3 the CDR L3 stop mutagenic oligonucleotide used to insert a TAA stopcodon at position 89 (Kabat numbering) within CDR L3 of the light chainwas 4D5LC3.stop (GCAACTTATTACTGTTAACAATCTTATACTACTC) (SEQ ID NO:106).The CDR H3 stop mutagenic oligonucleotide used to insert a TAA stopcodon at position 95 (Kabat numbering) within CDR H3 of the heavy chainof clone 1D8 was VH3.1D8.H3stop(GCCGTCTATTATTGTGCTCGTTAAGCCGGGTCCCGCTTGTTGTCG) (SEQ ID NO:107). The CDRH3 stop mutagenic oligonucleotide used to insert a TAA stop codon atposition 98 (Kabat numbering) within CDR H3 of the heavy chain of clone1E3 was 413Vh5SRo6(GAGGACACTGCCGTCTATTATTGTGCTCGTGAGGCCTCGTAACTGCCCCCCTACGTTATGGACTACTGGGGTCAAGGAACACTAGTC) (SEQ ID NO:108).

For clone 1F4 the CDR L1 stop mutagenic oligonucleotide used to insert aTAA stop codon at position 27 (Kabat numbering) within CDR L1 of thelight chain was CLC.L1stop (CATCACCTGCCGTGCCAGTTAATCCGTGTCCAGCGCTGTAG)(SEQ ID NO:109). For clone 1F4 the CDR L2 stop mutagenic oligonucleotideused to insert a TAA stop codon at position 52 (Kabat numbering) withinCDR L2 of the light chain was CLC.L2stop(CTTCTGATTTACTCGGCATAAAGCCTCTACTCTGGAGTC) (SEQ ID NO:110). For clone 1F4the CDR L3 stop mutagenic oligonucleotide used to insert a TAA stopcodon at position 90 (Kabat numbering) within CDR L3 of the light chainwas CLC4.1F4.L3stop (GCAACTTATTACTGTCAGTAATATTATTATTATTCTCCG) (SEQ IDNO:111). The CDR H3 stop mutagenic oligonucleotide used to insert a TAAstop codon at position 94 (Kabat numbering) within CDR H3 of the heavychain of clone 1F4 was CLC4.1F4.H3stop(GCCGTCTATTATTGTGCTTAAGGTTACGTTTGGAAAGGTG) (SEQ ID NO: 112).

B) Affinity Maturation Library Generation

A total of ten affinity maturation libraries were generated for eachclone (1F4, 1D8, and 1E3). All libraries were generated by Kunkelmutagenesis (see Kunkel, Proc. Natl. Acad. Sci. USA 82:488 (1985)). Inthe case of soft randomization, degenerate oligonucleotides weresynthesized such that the wild-type residue would be retained 50% of thetime and 50% of the time one of the remaining 19 amino acids would beencoded. To achieve soft randomization, oligonucleotides were designedsuch that certain nucleotide positions were occupied 70% of the timewith the indicated base and 10% of the time occupied by one of the threeother bases (Gallop et al., J. Med. Chem. 37:1233 (1994)). For thoseoligonucleotides that follow where such soft randomization was includedat a particular base, the presence of soft randomization is indicated bythe presence of a number at that base position. The number “5” indicatesthat the base adenine is present 70% of the time at that position, whilethe bases guanine, cytosine, and thymine are each present 10% of thetime. Similarly, the number “6” refers to guanine, “7” to cytosine, and“8” to thymine, where in each case, each of the other three bases ispresent only 10% of the time. In the case of hard randomization,degenerate oligonucleotides were synthesized such that amino aciddiversity found at certain positions within natural human antibodieswould be allowed. In this case degenerate codons were used where theletter “R” encodes for guanine or adenine, “Y” encodes for thymine orcytosine, “M” encodes for adenine or cytosine, “K” encodes for guanineor thymine, “S” encodes for guanine or cytosine, “W” encodes for adenineor thymine, “H” encodes for adenine, cytosine, or thymine, “B” encodesfor guanine, thymine, or cytosine, “V” encodes for guanine, cytosine, oradenine, “D” encodes for guanine, adenine, or thymine, and “N” encodesfor guanine, adenine, cytosine, or thymine.

Ten libraries were generated for 1F4 and designated L1, L2, L3,L1/L2/L3, H3, L3/H3, L1/H2, L2/H1, H2/H3, and L3/H1/H2. The 1F4 L1library had positions 28-33 (Kabat numbering) of the light chain hardrandomized to allow for amino acid diversity found at these positionswithin natural human antibodies. The L1 mutagenic oligonucleotidesF111-L1 (ACCTGCCGTGCCAGTCAGRDTRKTRVWANWTHTGTAGCCTGGTATCAACAGAAAC) (SEQID NO:113) and F202-L1 (ACCTGCCGTGCCAGTCAGRDTRKTRVWANWTHTCTGGCCTGGTATCAACAGAAAC) (SEQ ID NO:114) were mixed at a 1:2 ratioresulting in the “L1 oligo mix” and combined with 20 μg of Kunkel DNA ofthe 1F4 L1 stop template (described in Example 2A) to generate thelibrary by Kunkel mutagenesis.

The 1F4 L2 library had positions 50, 53, and 55 (Kabat numbering) of thelight chain hard randomized to allow for amino acid diversity found atthese positions within natural human antibodies. The L2 mutagenicoligonucleotides F201-L2 (CCGAAGCTTCTGATTTACKBGGCATCCAVCCTCTACTCTGGAGTCCCT) (SEQ ID NO:115) and F203-L2(CCGAAGCTTCTGATTTACKBGGCATCCAVCCTCGMATCTGGAGTCCCTTCTCGC) (SEQ ID NO:116)were mixed at a 1:1 ratio resulting in the “L2 oligo mix” and combinedwith 20 μg of Kunkel DNA of the 1F4 L2 stop template (described inExample 2A) to generate the library by Kunkel mutagenesis.

The 1F4 L3 library had positions 91-96 (Kabat numbering) of the lightchain either hard randomized to allow for amino acid diversity found atthese positions within natural human antibodies or soft randomized. Themutagenic oligonucleotides F133a (GCAACTTATTACTGTCAGCAATMTDMCRVTNHTCCTYKGACGTTCGGACAGGGTACC) (SEQ ID NO: 117),F133b (GCAACTTATTACTGTCAGCAATMTDMCRVTNHTC CTTWTACGTTCGGACAGGGTACC) (SEQID NO: 118), F133c (GCAACTTATTACTGTCAGCAASRTDMCRVTNHTCCTYKGACGTTCGGACAGGGTACC) (SEQ ID NO:119), F133d(GCAACTTATTACTGTCAGCAASRTDMCRVTNHTCCTT WTACGTTCGGACAGGGTACC) (SEQ IDNO:120) were mixed at a 1:1:1:1 ratio resulting in the “L3 hard oligomix”. The mutagenic oligonucleotides F563-L3soft1(ACTTATTACTGTCAGCAA878857577577CCT777ACGTTCGGACAGGGTACC) (SEQ IDNO:121), F564-L3soft2 (ACTTATTACTGTCAGCAA878857577577CCTTWTACGTTCGGACAGGGTACC) (SEQ ID NO:122), and F565-L3soft3 (ACTTATTACTGTCAGCAA878857577577CCTYKGACGTTCGGACAGGGTACC) (SEQ ID NO:123) were mixed at a1:0.5:1 ratio resulting in the “L3 soft oligo mix”. The “L3 hard oligomix”, the “L3 soft oligo mix”, and the mutagenic oligonucleotide 1F4.L3soft (GCAACTTATTACTGTCAG CAA857857857857878CCG788ACGTTCGGACAGGGTACCAAG)(SEQ ID NO:124) were then mixed at a 1:1:1 ratio resulting in the “L3total oligo mix” and combined with 20 μg of Kunkel DNA of the 1F4 L3stop template (described in Example 2A) to generate the library byKunkel mutagenesis.

The 1F4 H3 library had positions 95, 97, 99, 100, and 100a (Kabatnumbering) of the heavy chain soft randomized. Mutagenic oligonucleotideCLC.1F4.H3soft(GACACTGCCGTCTATTATTGTGCTCGC668TAC688TGG555668678ATGGACTACTGGGGTCAAGGAACC) (SEQ ID NO:125) was combined with 20 μg of Kunkel DNA ofthe 1F4 H3 stop template (described in Example 2A) to generate thelibrary by Kunkel mutagenesis.

The 1F4 L3/H3 library had positions 91-96 (Kabat numbering) of the lightchain either hard randomized to allow for amino acid diversity found atthese positions within natural human antibodies or soft randomized. Italso had positions 95, 97, 99, 100, and 100a (Kabat numbering) of theheavy chain soft randomized. The “L3 total oligo mix” and the mutagenicoligonucleotide CLC.1F4.H3soft (SEQ ID NO:126) described above weremixed at a 1:1 ratio and combined with 20 μg of Kunkel DNA of the 1F4L3/H3 stop template (described in Example 2A) to generate the library byKunkel mutagenesis.

The 1F4 L1/H2 library had positions 28-33 (Kabat numbering) of the lightchain hard randomized to allow for amino acid diversity found at thesepositions within natural human antibodies. It also had positions 50, 52,53, 54, and 58 (Kabat numbering) of the heavy chain soft randomized. The“L1 oligo mix” described above and the mutagenic oligonucleotideCLC4.1F4.H2soft (GGTAAGGGCCTGGAATGGGTTGCA878ATT857TCT857857AGCTATACT878TATGCCGATAGCGTCAAGGGCCG) (SEQ ID NO:126) were mixed at a 1:1ratio and combined with 20 μg of Kunkel DNA of the 1F4 L1 stop template(described in Example 2A) to generate the library by Kunkel mutagenesis.

The 1F4 L2/H1 library had positions 50, 53, and 55 (Kabat numbering) ofthe light chain hard randomized to allow for amino acid diversity foundat these positions within natural human antibodies. It also hadpositions 30-33 (Kabat numbering) of the heavy chain soft randomized.The “L2 oligo mix” described above and the mutagenic oligonucleotideCLC4.1F4.H1soft (GCAGCTTCTGGCTTCAACTTT857878857857ATGCACTGGGTGCGTCAGGCC)(SEQ ID NO:127) were mixed at a 1:1 ratio and combined with 20 μg ofKunkel DNA of the 1F4 L2 stop template (described in Example 2A) togenerate the library by Kunkel mutagenesis.

The 1F4 H2/H3 library had positions 50, 52, 53, 54, 58, 95, 97, 99, 100,and 100a (Kabat numbering) of the heavy chain soft randomized. Mutagenicoligonucleotides CLC4.1F4.H2soft (SEQ ID NO:126) and CLC4.1F4.H3soft(SEQ ID NO:125) described above were mixed at a 1:1 ratio and combinedwith 20 μg of Kunkel DNA of the 1F4 H3 stop template (described inExample 2A) to generate the library by Kunkel mutagenesis.

The 1F4 L1/L2/L3 library had positions 28-33, 50, 53, and 55 (Kabatnumbering) of the light chain hard randomized to allow for amino aciddiversity found at these positions within natural human antibodies. Italso had positions 91-96 (Kabat numbering) of the light chain eitherhard randomized to allow for amino acid diversity found at thesepositions within natural human antibodies or soft randomized. The “L1oligo mix”, the “L2 oligo mix”, and the “L3 total oligo mix” were mixedat a 1:1:1 ratio and combined with 20 μg of Kunkel DNA of the 1F4 L3stop template (described in Example 2A) to generate the library byKunkel mutagenesis.

The 1F4 L3/H1/H2 library had positions 91-96 (Kabat numbering) of thelight chain either hard randomized to allow for amino acid diversityfound at these positions within natural human antibodies or softrandomized. It also had positions 30-33, 50, 52, 53, 54, and 58 (Kabatnumbering) of the heavy chain soft randomized. The “L3 total oligo mix”,CLC4.1F4.H1soft (SEQ ID NO:127) and CLC4.1F4.H2soft (SEQ ID NO:126)described above were mixed at a 1:1:1 ratio and combined with 20 μg ofKunkel DNA of the 1F4 L3 stop template (described in Example 2A) togenerate the library by Kunkel mutagenesis.

Ten libraries were generated for 1D8 and designated L1, L2, L3,L1/L2/L3, H3, L3/H3, L1/H2, L2/H1, H2/H3, and L3/H1/H2. The 1D8 L1library had positions 28-33 (Kabat numbering) of the light chain hardrandomized to allow for amino acid diversity found at these positionswithin natural human antibodies. The L1 mutagenic oligonucleotidesF111-L1 (SEQ ID NO:113) and F202-L1 (SEQ ID NO:114) described above weremixed at a 1:2 ratio resulting in the “L1 oligo mix” and combined with20 μg of Kunkel DNA of the 1D8 L1 stop template (described in Example2A) to generate the library by Kunkel mutagenesis.

The 1D8 L2 library had positions 50, 53, and 55 (Kabat numbering) of thelight chain hard randomized to allow for amino acid diversity found atthese positions within natural human antibodies. The L2 mutagenicoligonucleotides F201-L2 (SEQ ID NO:115) and F203-L2 (SEQ ID NO:116)described above were mixed at a 1:1 ratio resulting in the “L2 oligomix” and combined with 20 μg of Kunkel DNA of the 1D8 L2 stop template(described in Example 2A) to generate the library by Kunkel mutagenesis.

The 1D8 L3 library had positions 91-94 and 96 (Kabat numbering) of thelight chain either hard randomized to allow for amino acid diversityfound at these positions within natural human antibodies or softrandomized. The mutagenic oligonucleotides F133a (SEQ ID NO:117), F133b(SEQ ID NO:118), F133c (SEQ ID NO:119), and F133d (SEQ ID NO:120)described above were mixed at a 1:1:1:1 ratio resulting in the “L3 hardoligo mix”. The mutagenic oligonucleotides F563-L3soft1 (SEQ ID NO:121),F564-L3soft2 (SEQ ID NO:122), and F565-L3soft3 (SEQ ID NO:123) describedabove were mixed at a 1:0.5:1 ratio resulting in the “L3 soft oligomix”. The “L3 hard oligo mix” and the “L3 soft oligo mix” were thenmixed at a 1:1 ratio and combined with 20 μg of Kunkel DNA of the 1D8 L3stop template (described in Example 2A) to generate the library byKunkel mutagenesis.

The 1D8 H3 library had positions 96-100c (Kabat numbering) of the heavychain soft randomized. Mutagenic oligonucleotide VH3.1D8.H3soft(GCCGTCTATTATTGTGCTCGTGAG678668878565788788878688ATGGACTACTGGGGTCAAGGAACC) (SEQ ID NO:128)was combined with 20 μg of Kunkel DNA of the 1D8 H3 stop template(described in Example 2A) to generate the library by Kunkel mutagenesis.

The 1D8 L3/H3 library had positions 91-94 and 96 (Kabat numbering) ofthe light chain either hard randomized to allow for amino acid diversityfound at these positions within natural human antibodies or softrandomized. It also had positions 96-100c (Kabat numbering) of the heavychain soft randomized. The “L3 soft oligo mix”, the “L3 hard oligo mix”,and the mutagenic oligonucleotide VH3.1D8.H3soft (SEQ ID NO:128)described above were mixed at a 0.5:0.5:1 ratio and combined with 20 μgof Kunkel DNA of the 1D8 L3/H3 stop template (described in Example 2A)to generate the library by Kunkel mutagenesis.

The 1D8 L1/H2 library had positions 28-33 (Kabat numbering) of the lightchain hard randomized to allow for amino acid diversity found at thesepositions within natural human antibodies. It also had positions 50, 52,53, 54, and 58 (Kabat numbering) of the heavy chain soft randomized. The“L1 oligo mix” described above and the mutagenic oligonucleotideVH3.1D8.H2soft (GGTAAGGGCCTGGAATGGGTTGCT668ATT878CCT857668GGTTATACT657TATGCCGATAGCGTCAAGGGCCG) (SEQ ID NO:129) were mixed at a 1:1ratio and combined with 20 μg of Kunkel DNA of the 1D8 L1 stop template(described in Example 2A) to generate the library by Kunkel mutagenesis.

The 1D8 L2/H1 library had positions 50, 53, and 55 (Kabat numbering) ofthe light chain hard randomized to allow for amino acid diversity foundat these positions within natural human antibodies. It also hadpositions 30-33 (Kabat numbering) of the heavy chain soft randomized.The “L2 oligo mix” described above and the mutagenic oligonucleotideVH3.1D8.H1soft (GCAGCTTCTGGCTTCACCTTC577657857657ATTCACTGGGTGCGTCAGGCC)(SEQ ID NO:130) were mixed at a 1:1 ratio and combined with 20 μg ofKunkel DNA of the 1D8 L2 stop template (described in Example 2A) togenerate the library by Kunkel mutagenesis.

The 1D8 H2/H3 library had positions 50, 52, 53, 54, 58, and 96-100c(Kabat numbering) of the heavy chain soft randomized. Mutagenicoligonucleotides VH3.1D8.H2soft (SEQ ID NO:129) and VH3.1D8.H3soft (SEQID NO:128) described above were mixed at a 1:1 ratio and combined with20 μg of Kunkel DNA of the 1D8 H3 stop template (described in Example2A) to generate the library by Kunkel mutagenesis.

The 1D8 L1/L2/L3 library had positions 28-33, 50, 53, and 55 (Kabatnumbering) of the light chain hard randomized to allow for amino aciddiversity found at these positions within natural human antibodies. Italso had positions 91-94 and 96 (Kabat numbering) of the light chaineither hard randomized to allow for amino acid diversity found at thesepositions within natural human antibodies or soft randomized. The “L1oligo mix”, the “L2 oligo mix”, the “L3 hard oligo mix”, and the “L3soft oligo mix” were mixed at a 1:1:0.5:0.5 ratio and combined with 20μg of Kunkel DNA of the 1D8 L3 stop template (described in Example 2A)to generate the library by Kunkel mutagenesis.

The 1D8 L3/H1/H2 library had positions 91-94 and 96 (Kabat numbering) ofthe light chain either hard randomized to allow for amino acid diversityfound at these positions within natural human antibodies or softrandomized. It also had positions 30-33, 50, 52, 53, 54, and 58 (Kabatnumbering) of the heavy chain soft randomized. The “L3 hard oligo mix”,the “L3 soft oligo mix”, VH3.1D8.H1soft (SEQ ID NO:130) andVH3.1D8.H2soft (SEQ ID NO:129) described above were mixed at a0.5:0.5:1:1 ratio and combined with 20 μg of Kunkel DNA of the 1D8 L3stop template (described in Example 2A) to generate the library byKunkel mutagenesis.

Ten libraries were generated for 1E3 and designated L1, L2, L3,L1/L2/L3, H3, L3/H3, L1/H2, L2/H1, H2/H3, and L3/H1/H2. The 1E3 L1library had positions 28-33 (Kabat numbering) of the light chain hardrandomized to allow for amino acid diversity found at these positionswithin natural human antibodies. The L1 mutagenic oligonucleotidesF111-L1 (SEQ ID NO:113) and F202-L1 (SEQ ID NO:114) described above weremixed at a 1:2 ratio resulting in the “L1 oligo mix” and combined with20 μg of Kunkel DNA of the 1E3 L1 stop template (described in Example2A) to generate the library by Kunkel mutagenesis.

The 1E3 L2 library had positions 50, 53, and 55 (Kabat numbering) of thelight chain hard randomized to allow for amino acid diversity found atthese positions within natural human antibodies. The L2 mutagenicoligonucleotides F201-L2 (SEQ ID NO:115) and F203-L2 (SEQ ID NO:116)described above were mixed at a 1:1 ratio resulting in the “L2 oligomix” and combined with 20 μg of Kunkel DNA of the 1E3 L2 stop template(described in Example 2A) to generate the library by Kunkel mutagenesis.

The 1E3 L3 library had positions 91-94 and 96 (Kabat numbering) of thelight chain either hard randomized to allow for amino acid diversityfound at these positions within natural human antibodies or softrandomized. The mutagenic oligonucleotides F133a (SEQ ID NO:117), F133b(SEQ ID NO:118), F133c (SEQ ID NO:119), and F133d (SEQ ID NO:120)described above were mixed at a 1:1:1:1 ratio resulting in the “L3 hardoligo mix”. The mutagenic oligonucleotides F563-L3soft1 (SEQ ID NO:121),F564-L3soft2 (SEQ ID NO:122), and F565-L3soft3 (SEQ ID NO:123) describedabove were mixed at a 1:0.5:1 ratio resulting in the “L3 soft oligomix”. The “L3 hard oligo mix” and the “L3 soft oligo mix” were thenmixed at a 1:1 ratio and combined with 20 μg of Kunkel DNA of the 1E3 L3stop template (described in Example 2A) to generate the library byKunkel mutagenesis.

The 1E3 H3 library had positions 95, 97, 98, 99, and 100a (Kabatnumbering) of the heavy chain soft randomized. Mutagenic oligonucleotideVH4.1E3.H3soft(GACACTGCCGTCTATTATTGTGCTCGT577TGG788788565TGG688ATGGACTACTGGGGTCAAGGAACCCTG) (SEQ ID NO:131) was combined with 20 μg of Kunkel DNA ofthe 1E3 H3 stop template (described in Example 2A) to generate thelibrary by Kunkel mutagenesis.

The 1E3 L3/H3 library had positions 91-94 and 96 (Kabat numbering) ofthe light chain either hard randomized to allow for amino acid diversityfound at these positions within natural human antibodies or softrandomized. It also had positions 95, 97, 98, 99, and 100a (Kabatnumbering) of the heavy chain soft randomized. The “L3 soft oligo mix”,the “L3 hard oligo mix”, and the mutagenic oligonucleotideVH4.1E3.H3soft (SEQ ID NO:131) described above were mixed at a 0.5:0.5:1ratio and combined with 20 μg of Kunkel DNA of the 1E3 L3/H3 stoptemplate (described in Example 2A) to generate the library by Kunkelmutagenesis.

The 1E3 L1/H2 library had positions 28-33 (Kabat numbering) of the lightchain hard randomized to allow for amino acid diversity found at thesepositions within natural human antibodies. It also had positions 50, 52,53, 54, and 58 (Kabat numbering) of the heavy chain soft randomized. The“L1 oligo mix” described above and the mutagenic oligonucleotide VH4.1E3.H2soft (GGTAAGGGCCTGGAATGGGTTGCT878ATT577CCT878878GGTTCTACT657TATGCCGATAGCGTCAAGGGCCG) (SEQ ID NO:132) were mixed at a 1:1 ratioand combined with 20 μg of Kunkel DNA of the 1E3 L1 stop template(described in Example 2A) to generate the library by Kunkel mutagenesis.

The 1E3 L2/H1 library had positions 50, 53, and 55 (Kabat numbering) ofthe light chain hard randomized to allow for amino acid diversity foundat these positions within natural human antibodies. It also hadpositions 30-33 (Kabat numbering) of the heavy chain soft randomized.The “L2 oligo mix” described above and the mutagenic oligonucleotideVH4.1E3.H1soft (GCAGCTTCTGGCTTCACCTTC878558577857ATTAGCTGGGTGCGTCAGGCC)(SEQ ID NO:133) were mixed at a 1:1 ratio and combined with 20 μg ofKunkel DNA of the 1E3 L2 stop template (described in Example 2A) togenerate the library by Kunkel mutagenesis.

The 1E3 H2/H3 library had positions 50, 52, 53, 54, 58, 95, 97, 98, 99,and 100a (Kabat numbering) of the heavy chain soft randomized. Mutagenicoligonucleotides VH4.1E3.H2soft (SEQ ID NO:132) and VH4.1E3.H3soft (SEQID NO:133) described above were mixed at a 1:1 ratio and combined with20 μg of Kunkel DNA of the 1E3 H3 stop template (described in Example2A) to generate the library by Kunkel mutagenesis.

The 1E3 L1/L2/L3 library had positions 28-33, 50, 53, and 55 (Kabatnumbering) of the light chain hard randomized to allow for amino aciddiversity found at these positions within natural human antibodies. Italso had positions 91-94 and 96 (Kabat numbering) of the light chaineither hard randomized to allow for amino acid diversity found at thesepositions within natural human antibodies or soft randomized. The “L1oligo mix”, the “L2 oligo mix”, the “L3 hard oligo mix”, and the “L3soft oligo mix” were mixed at a 1:1:0.5:0.5 ratio and combined with 20μg of Kunkel DNA of the 1E3 L3 stop template (described in Example 2A)to generate the library by Kunkel mutagenesis.

The 1E3 L3/H1/H2 library had positions 91-94 and 96 (Kabat numbering) ofthe light chain either hard randomized to allow for amino acid diversityfound at these positions within natural human antibodies or softrandomized. It also had positions 30-33, 50, 52, 53, 54, and 58 (Kabatnumbering) of the heavy chain soft randomized. The “L3 hard oligo mix”,the “L3 soft oligo mix”, VH4.1E3.H1soft (SEQ ID NO:133) andVH4.1E3.H2soft (SEQ ID NO:132) described above were mixed at a0.5:0.5:1:1 ratio and combined with 20 μg of Kunkel DNA of the 1E3 L3stop template (described in Example 2A) to generate the library byKunkel mutagenesis.

The mutagenesis reactions were electroporated into electrocompetentXL1-Blue (Agilent) E. coli and recovered in 25 mL of SOC medium for 45minutes at 37° C. with shaking. Twenty microliters were removed andten-fold serial dilutions were plated onto solid agar plates containingcarbenicillin and grown overnight at 37° C. to determine the librarysize. The remaining culture was transferred to 500 mL of 2YT brothcontaining 50 μg/mL carbenicillin and 1×10¹⁰ phage/mL M13K07 helperphage. The cells were infected at 37° C. for one hour with shaking. 50μg/mL of kanamycin was added and the cultures were grown for anotherseven hours at 37° C. with shaking. The temperature was then shifted to30° C. and the cultures were grown for another 22 hours. The librarieseach contained at least ˜9.5×10⁹ colony forming units (CFUs). The phagewere purified from the culture supernatant by two rounds ofprecipitation with 1/5 volume of 20% polyethylene glycol (PEG)/2.5MNaCl.

C) Affinity Maturation Library Sorting

The 1F4, 1D8, and 1E3 affinity maturation libraries underwent fourrounds of sorting. Each of the ten sub-libraries were sorted in parallelfor the first round and then pooled for sorts two through four. Thefirst round was plate-based sorting with linear diubiquitin immobilizedon a 96-well Maxisorb immunoplate (NUNC). Plates were coated overnightat 4° C. with 5 μg/mL linear diubiquitin (Boston Biochem) in 50 mMsodium carbonate buffer, pH 9.6. The coated plates were blocked with 200μL/well of 2.5% milk in PBS containing 0.05% Tween 20 (PBST) for onehour at 25° C. with shaking. The phage libraries were diluted to anOD=2.0 in 2.5% milk in PBST and 30 μg/mL of K63-linked polyubiquitin 2-7(Boston Biochem) was added for counterselection. After one hour, theblocking buffer was dumped off of the plate and 100 μL/well of the phagewas added and incubated at 25° C. for three hours with shaking. Afterbinding, the plate was washed 20 times with PBST by manually filling thewells and dumping off the buffer between washes. Phage were eluted with150 μL/well of 50 mM HCl/500 mM KCl for 30 minutes at 25° C. withshaking.

The elution was neutralized with 150 μL/well of 1 M Tris, pH 7.5 andsubsequently propagated in XL1-Blue (Agilent) E. coli with the additionof M13K07 helper phage. Amplified phage were used for additional roundsof selection against linear diubiquitin in plate-based sorting.Solution-based sorting was not possible because biotinylation of lineardiubiquitin interfered with Fab binding. Stringency of the later sortswas increased in three ways: by adding 30 μg/mL soluble monoubiquitin,K11-linked polyubiquitin, K48-linked polyubiquitin 2-7, and K63-linkedpolyubiquitin 2-7 to the phage for counterselection; by increasing thenumber and duration of plate washes; and by decreasing the amount ofphage used and the duration of phage binding. The second sort was doneexactly as the first sort, except that the soluble ubiquitins added wasexpanded to include the above listed chains, the amount of phage usedwas OD₂₆₈=1.0, the duration of phage binding was decreased to 2 hours,and the number of plate washes was increased to 30. The third sort wasdone exactly as the second sort, except that the duration of phagebinding was reduced to 1.5 hours and the number of plate washes wasincreased to 40 followed by four additional washes of 15 minutes eachwith shaking at 25° C. with five quick washes in between each 15 minutewash. The fourth sort was done exactly as the third sort, except thatthe amount of phage used was reduced to OD₂₆₈=0.5, the duration of phagebinding was reduced to 1 hour, and the washes included 40 quick washesfollowed by four 15 minute washes with shaking at 37° C. Enrichment wascalculated for rounds two through four by comparing the number of phagerecovered with linear diubiquitin compared to an uncoated well.Enrichment was observed in rounds two through four for all threelibraries (see Table 3).

TABLE 3 Library Round 2 Round 3 Round 4 1F4 AM 143X  500X 5000X 1D8 AM850X 1400X 5500X 1E3 AM  45X  167X  60X

After four rounds of sorting 96 individual clones were picked from the1F4 third round sort, 1D8 second round sort, 1D8 third round sort, 1D8fourth round sort, and 1E3 third round sort and grown up in 96-wellformat in 1 mL of 2YT broth containing 50 μg/mL carbenicillin and 1×10¹⁰phage/mL M13K07 helper phage. Supernatants from those cultures were usedin high-throughput phage spot ELISAs for binding to linear diubiquitin(Boston Biochem), monoubiquitin (Boston Biochem), K11-linked diubiquitin(Genentech), K48-linked diubiquitin (Boston Biochem), K63-linkeddiubiquitin (Boston Biochem), an anti-gD antibody (Genentech), or anuncoated well (as described in Example 1B). From the 1F4 third roundsort all clones were very weak linear diubiquitin binders showing OD₄₅₀of less than 0.4 and therefore were not pursued. From the 1D8 secondround sort 93 of the clones were linear diubiquitin-specific, butsequencing revealed that all were the parental wild-type 1D8 sequence.From the 1D8 third round sort 48 of the clones were lineardiubiquitin-specific, but sequencing revealed that all but two were theparental wild-type 1D8 sequence. The two non-parental clones 1D8.3C2(SEQ ID NOs: 33 and 36) and 1D8.3F8 (SEQ ID NOs: 34 and 37) (see FIGS.5A and 5B) were tested by phage IC₅₀ ELISA as in example 1D anddemonstrated to have IC₅₀s in the low μM range for linear diubiquitin,only slightly improved over 1D8 (see FIG. 6 ). From the 1D8 fourth roundsort 94 of the clones showed strong binding to both linear diubiquitinand K63-linked diubiquitin (OD₄₅₀ greater than 1.0 for linear, OD₄₅₀greater than 0.5 for K63) and therefore were not pursued. Only oneclone, 1D8.4F5 (SEQ ID NOs: 35 and 38) (see FIGS. 5A and 5B) showedstrong linear diubiquitin binding with little K63-linked diubiquitinbinding (OD₄₅₀˜1.3, OD₄₅₀˜0.1 for K63). The phage IC₅₀ for 1D8.4F5 wasalso measured by ELISA as in example 1D and determined to be ˜2 μM, onlyslightly better than the parental clone 1D8 (see FIG. 6 ). From the 1E3third round sort 33 of the clones were linear diubiquitin-specific,however they were all very weak binders (OD₄₅₀ of less than 0.5) andtherefore were not pursued. An additional 27 clones showed strongerbinding to linear diubiquitin (OD₄₅₀ of greater than 0.5 but less than1.0) however they also showed increased binding to K63-linkeddiubiquitin (OD₄₅₀ of greater than 0.1) and therefore were not pursued.

Example 3—Second Affinity Maturation of 1E3

A) Stop Template Generation

A TAA stop codon was inserted separately into either CDR L1, CDR L2, CDRL3, CDR H1, CDR H2, and CDR H3 (resulting in L1, L2, L3, H1, H2, and H3stop templates, respectively) for library synthesis using Kunkelmutagenesis. Stop codons force diversity within a particular CDR loop byrequiring repair of the stop in order to get full length Fab expressionand display on phage. The stop codon mutagenic oligonucleotides listedbelow were combined with 1 μg of 1E3 monovalent phagemid Kunkel DNA. Theresulting monovalent Fab phagemid stop templates were used for affinitymaturation library generation.

The mutagenic oligonucleotide used to insert a TAA stop codon atposition 31 (Kabat numbering) within CDR L1 of the 1E3 light chain wasE3.L1stop (GCCAGTCAGGATGTG TCCTAAGCTGTAGCCTGGTATCAAC) (SEQ ID NO:134).The mutagenic oligonucleotide used to insert a TAA stop codon atposition 53 (Kabat numbering) within CDR L2 of the 1E3 light chain wasE3.L2stop (CTGATTTACTCGGCATCCTAACTCTACTCTGGAGTCCCTTC) (SEQ ID NO:135).The mutagenic oligonucleotide used to insert a TAA stop codon atposition 93 (Kabat numbering) within CDR L3 of the 1E3 light chain wasE3.L3stop (CTTATTACT GTCAGCAATCTTATTAAACTCCTCCCACGTTCGGACAG) (SEQ IDNO:136). The mutagenic oligonucleotide used to insert a TAA stop codonat position 32 (Kabat numbering) within CDR H1 of the 1E3 light chainwas E3.H1stop (GGCTTCACCTTC AGTAATTAATATATTAGCTGGGTGCGTC) (SEQ IDNO:137). The mutagenic oligonucleotide used to insert a TAA stop codonat position 54 (Kabat numbering) within CDR H2 of the 1E3 light chainwas E3.H2stop (GTTGCTTCTATTACTCCTTAAAGCGGTTCTACTGACTATG) (SEQ IDNO:138). The mutagenic oligonucleotide used to insert a TAA stop codonat position 99 (Kabat numbering) within CDR H3 of the 1E3 light chainwas E3.H3stop (GCTCGTACCTGGTTGCTCTAATGGGTTATGGACTACTGG) (SEQ ID NO:139).

B) Single Position NNK Library Generation

Since the first attempt at affinity maturation of 1F4 and 1D8 resultedin only modest improvements in affinity with IC₅₀s still in the low μMrange, clone 1E3 which had a starting IC₅₀ of 80 nM was focused on. Thefirst attempt at 1E3 affinity maturation produced many clones thatshowed strong binding to both linear and K63-linked diubiquitin.Therefore a different approach was taken to minimize K63-linkeddiubiquitin binding by limiting the number of mutations incorporatedinto single clones. Single position NNK randomization was used toincorporate a single amino acid change into one CDR at a time. Sixsingle CDR libraries designated L1, L2, L3, H1, H2, and H3 weregenerated where a single residue in any one clone was allowed to retainthe wild-type residue or to change to any one of the other 19 aminoacids.

The L1 library had positions 28-34 (Kabat numbering) of the light chainhard randomized individually using the NNK codon to allow for all 20amino acids. The L1 mutagenic oligonucleotides

-   -   E3.L1.1 (CATCACCTGCCGTGCCAGTCAGNNKGTGTCCACTGCTG        TAGCCTGGTATCAACAGAAACCAGG) (SEQ ID NO:140),    -   E3.L1.2 (CATCACCTGCCGTGCCAGTCAGGATNNKTCCACTGCTGTAGCCTGGTATC        AACAGAAACCAGG) (SEQ ID NO:141),    -   E3.L1.3 (CATCACCTGCCGTGCCAGTCAGGATGTGNNKACTGCTGTAGCCTGGTATCA        ACAGAAACCAGG) (SEQ ID NO:142),    -   E3.L1.4 (CATCACCTGCCGTGCCAGTCAGGATGTGTCCNNKGCTGTAGCCTGGTATCAA        CAGAAACCAGG) (SEQ ID NO:143),    -   E3.L1.5 (CATCACCTGCCGTGCCAGTCAGGATGTGTCCACTNNKGTAGCCTGGTATCA        ACAGAAACCAGG) (SEQ ID NO:144),    -   E3.L1.6 (CATCACCTGCCGTGCCAGTCAGGATGTGTCCACTGCTNNKGCCTGGTATCAA        CAGAAACCAGG) (SEQ ID NO:145), and    -   E3.L1.7 (CATCACCTGCCGTGCCAGTCAGGATGTGTCCACTGCTGTANNKTGGTATCAA        CAGAAACCAGG) (SEQ ID NO:146) were mixed at a 1:1:1:1:1:1:1 ratio        and combined with 20 μg of Kunkel DNA of the 1E3 L1 stop        template (described in Example 3A) to generate the library by        Kunkel mutagenesis.

The L2 library had positions 50-56 (Kabat numbering) of the light chainhard randomized individually using the NNK codon to allow for all 20amino acids. The L2 mutagenic oligonucleotides

-   -   E3.L2.1 (GCTCCGAAGCTTCTGATTTACNNKGCATCCTTCCTCTACTCTG        GAGTCCCTTCTCGCTTCTCTG) (SEQ ID NO:147),    -   E3.L2.2 (GCTCCGAAGCTTCTGATTTACTCGNNKTCCTTCCTCTACTCTGGAGTCCCTT        CTCGCTTCTCTG) (SEQ ID NO:148),    -   E3.L2.3 (GCTCCGAAGCTTCTGATTTACTCGGCANNKTTCCTCTACTCTGGAGTCCCTT        CTCGCTTCTCTG) (SEQ ID NO:149),    -   E3.L2.4 (GCTCCGAAGCTTCTGATTTACTCGGCATCCNNKCTCTACTCTGGAGTCCCTT        CTCGCTTCTCTG) (SEQ ID NO:150),    -   E3.L2.5 (GCTCCGAAGCTTCTGATTTACTCGGCATCCTTCNNKTACTCTGGAGTCCCTT        CTCGCTTCTCTG) (SEQ ID NO:151),    -   E3.L2.6 (GCTCCGAAGCTTCTGATTTACTCGGCATCCTTCCTCNNKTCTGGAGTCCCTT        CTCGCTTCTCTG) (SEQ ID NO:152), and    -   E3.L2.7 (GCTCCGAAGCTTCTGATTTACTCGGCATCCTTCCTCTACNNKGGAGTCCCTT        CTCGCTTCTCTG) (SEQ ID NO:153) were mixed at a 1:1:1:1:1:1:1        ratio and combined with 20 μg of Kunkel DNA of the 1E3 L2 stop        template (described in Example 3A) to generate the library by        Kunkel mutagenesis.

The L3 library had positions 91-96 (Kabat numbering) of the light chainhard randomized individually using the NNK codon to allow for all 20amino acids. The L3 mutagenic oligonucleotides

-   -   E3.L3.1 (GCAACTTATTACTGTCAGCAANNKTATACTACTCCTCCCACG        TTCGGACAGGGTACCAAG) (SEQ ID NO:154),    -   E3.L3.2 (GCAACTTATTACTGTCAGCAATCTNNKACTACTCCTCCCACGTTCGGACA        GGGTACCAAG) (SEQ ID NO:155),    -   E3.L3.3 (GCAACTTATTACTGTCAGCAATCTTATNNKACTCCTCCCACGTTCGGACA        GGGTACCAAG) (SEQ ID NO:156),    -   E3.L3.4 (GCAACTTATTACTGTCAGCAATCTTATACTNNKCCTCCCACGTTCGGACA        GGGTACCAAG) (SEQ ID NO:157),    -   E3.L3.5 (GCAACTTATTACTGTCAGCAATCTTATACTACTNNKCCCACGTTCGGACA        GGGTACCAAG) (SEQ ID NO:158), and    -   E3.L3.6 (GCAACTTATTACTGTCAGCAATCTTATACTACTCCTNNKACGTTCGGACA        GGGTACCAAG) (SEQ ID NO:159) were mixed at a 1:1:1:1:1:1 ratio        and combined with 20 μg of Kunkel DNA of the 1E3 L3 stop        template (described in Example 3A) to generate the library by        Kunkel mutagenesis.

The H1 library had positions 30-35 (Kabat numbering) of the heavy chainhard randomized individually using the NNK codon to allow for all 20amino acids. The H1 mutagenic oligonucleotides

-   -   E3.H1.1 (GCAGCTTCTGGCTTCACCTTCNNKAATACTTATATTAGCT        GGGTGCGTCAGGCCCCG) (SEQ ID NO:160),    -   E3.H1.2 (GCAGCTTCTGGCTTCACCTTCAGTNNKACTTATATTAGCTGGGTGCG        TCAGGCCCCG) (SEQ ID NO:161),    -   E3.H1.3 (GCAGCTTCTGGCTTCACCTTCAGTAATNNKTATATTAGCTGGGTGCG        TCAGGCCCCG) (SEQ ID NO:162),    -   E3.H1.4 (GCAGCTTCTGGCTTCACCTTCAGTAATACTNNKATTAGCTGGGTGCG        TCAGGCCCCG) (SEQ ID NO:163),    -   E3.H1.5 (GCAGCTTCTGGCTTCACCTTCAGTAATACTTATNNKAGCTGGGTGCG        TCAGGCCCCG) (SEQ ID NO:164), and    -   E3.H1.6 (GCAGCTTCTGGCTTCACCTTCAGTAATACTTATATTNNKTGGGTGCG        TCAGGCCCCG) (SEQ ID NO:165) were mixed at a 1:1:1:1:1:1 ratio        and combined with 20 μg of Kunkel DNA of the 1E3 H1 stop        template (described in Example 3A) to generate the library by        Kunkel mutagenesis.

The H2 library had positions 49-58 (Kabat numbering) of the heavy chainhard randomized individually using the NNK codon to allow for all 20amino acids. The H2 mutagenic oligonucleotides

-   -   E3.H2.1 (GGTAAGGGCCTGGAATGGGTTNNKTCTATTACTCCTTCTAGCGGTTCTACTG        ACTATGCCGATAGCGTCAAGGGC) (SEQ ID NO:166),    -   E3.H2.2 (GGTAAGGGCCTGGAATGGGTTGCTNNKATTACTCCTTCTAGCGGTTCTACTG        ACTATGCCGATAGCGTCAAGGGC) (SEQ ID NO:167),    -   E3.H2.3 (GGTAAGGGCCTGGAATGGGTTGCTTCTNNKACTCCTTCTAGCGGTTCTACTG        ACTATGCCGATAGCGTCAAGGGC) (SEQ ID NO:168),    -   E3.H2.4 (GGTAAGGGCCTGGAATGGGTTGCTTCTATTNNKCCTTCTAGCGGTTCTACTG        ACTATGCCGATAGCGTCAAGGGC) (SEQ ID NO:169),    -   E3.H2.5 (GGTAAGGGCCTGGAATGGGTTGCTTCTATTACTNNKTCTAGCGGTTCTACTG        ACTATGCCGATAGCGTCAAGGGC) (SEQ ID NO:170),    -   E3.H2.6 (GGTAAGGGCCTGGAATGGGTTGCTTCTATTACTCCTNNKAGCGGTTCTACTG        ACTATGCCGATAGCGTCAAGGGC) (SEQ ID NO:171),    -   E3.H2.7 (GGTAAGGGCCTGGAATGGGTTGCTTCTATTACTCCTTCTNNKGGTTCTACTG        ACTATGCCGATAGCGTCAAGGGC) (SEQ ID NO:172),    -   E3.H2.8 (GGTAAGGGCCTGGAATGGGTTGCTTCTATTACTCCTTCTAGCNNKTCTACTG        ACTATGCCGATAGCGTCAAGGGC) (SEQ ID NO:173),    -   E3.H2.9 (GGTAAGGGCCTGGAATGGGTTGCTTCTATTACTCCTTCTAGCGGTNNKACTG        ACTATGCCGATAGCGTCAAGGGC) (SEQ ID NO:174),    -   E3.H2.10 (GGTAAGGGCCTGGAATGGGTTGCTTCTATTACTCCTTCTAGCGGTTCTNNKG        ACTATGCCGATAGCGTCAAGGGC) (SEQ ID NO:175), and    -   E3.H2.11 (GGTAAGGGCCTGGAATGGGTTGCTTCTATTACTCCTTCTAGCGGTTCTACT        NNKTATGCCGATAGCGTCAAGGGC) (SEQ ID NO:176) were mixed at a        1:1:1:1:1:1:1:1:1:1:1 ratio and combined with 20 μg of Kunkel        DNA of the 1E3 H2 stop template (described in Example 3A) to        generate the library by Kunkel mutagenesis.

The H3 library had positions 95-102 (Kabat numbering) of the heavy chainhard randomized individually using the NNK codon to allow for all 20amino acids. The H3 mutagenic oligonucleotides

-   -   E3.H3.1 (GCCGTCTATTATTGTGCTCGTNNKTGGTTGCTCCGGTGGGTTATGGACTAC        TGGGGTCAAGGAACCCTGGTC) (SEQ ID NO:177),    -   E3.H3.2 (GCCGTCTATTATTGTGCTCGTACCNNKTTGCTCCGGTGGGTTATGGACTAC        TGGGGTCAAGGAACCCTGGTC) (SEQ ID NO:178),    -   E3.H3.3 (GCCGTCTATTATTGTGCTCGTACCTGGNNKCTCCGGTGGGTTATGGACTAC        TGGGGTCAAGGAACCCTGGTC) (SEQ ID NO:179),    -   E3.H3.4 (GCCGTCTATTATTGTGCTCGTACCTGGTTGNNKCGGTGGGTTATGGACTAC        TGGGGTCAAGGAACCCTGGTC) (SEQ ID NO:180),    -   E3.H3.5 (GCCGTCTATTATTGTGCTCGTACCTGGTTGCTCNNKTGGGTTATGGACTAC        TGGGGTCAAGGAACCCTGGTC) (SEQ ID NO:181),    -   E3.H3.6 (GCCGTCTATTATTGTGCTCGTACCTGGTTGCTCCGGNNKGTTATGGACTAC        TGGGGTCAAGGAACCCTGGTC) (SEQ ID NO:182),    -   E3.H3.7 (GCCGTCTATTATTGTGCTCGTACCTGGTTGCTCCGGTGGNNKATGGACTAC        TGGGGTCAAGGAACCCTGGTC) (SEQ ID NO:183),    -   E3.H3.8 (GCCGTCTATTATTGTGCTCGTACCTGGTTGCTCCGGTGGGTTNNKGACTAC        TGGGGTCAAGGAACCCTGGTC) (SEQ ID NO:184),    -   E3.H3.9 (GCCGTCTATTATTGTGCTCGTACCTGGTTGCTCCGGTGGGTTATGNNKTAC        TGGGGTCAAGGAACCCTGGTC) (SEQ ID NO:185), and    -   E3.H3.10 (GCCGTCTATTATTGTGCTCGTACCTGGTTGCTCCGGTGGGTTATGGACNNKT        GGGGTCAAGGAACCCTGGTC) (SEQ ID NO:186) were mixed at a        1:1:1:1:1:1:1:1:1:1 ratio and combined with 20 μg of Kunkel DNA        of the 1E3 H3 stop template (described in Example 3A) to        generate the library by Kunkel mutagenesis.

The mutagenesis reactions were electroporated into electrocompetentXL1-Blue (Agilent) E. coli and recovered in 25 mL of SOC medium for 45minutes at 37° C. with shaking. Twenty microliters were removed andten-fold serial dilutions were plated onto solid agar plates containingcarbenicillin and grown overnight at 37° C. to determine the librarysize. The remaining culture was transferred to 500 mL of 2YT brothcontaining 50 μg/mL carbenicillin and 1×10¹⁰ phage/mL M13K07 helperphage. The cells were infected at 37° C. for one hour with shaking. 50μg/mL of kanamycin was added and the cultures were grown for anotherseven hours at 37° C. with shaking. The temperature was then shifted to30° C. and the cultures were grown for another 22 hours. The librarieseach contained at least ˜1.5×10¹⁰ colony forming units (CFUs). The phagewere purified from the culture supernatant by two rounds ofprecipitation with 1/5 volume of 20% polyethylene glycol (PEG)/2.5MNaCl.

Sixty-four individual clones from each of the six libraries weresequenced to make sure the amino acid diversity in the libraryaccurately reflected the design. The libraries all were as designed.

C) Affinity Maturation Library Sorting

The six CDR NNK affinity maturation libraries underwent three rounds ofsorting in parallel against either linear diubiquitin or K63-linkeddiubiquitin. Plates were coated overnight at 4° C. with 5 μg/mL lineardiubiquitin (Boston Biochem) or K63-linked diubiquitin (Boston Biochem)in 50 mM sodium carbonate buffer, pH 9.6. The coated plates were blockedwith 200 μL/well of 2.5% milk in PBS containing 0.05% Tween 20 (PBST)for one hour at 25° C. with shaking. The phage libraries were diluted toan OD=1.0 in 2.5% milk in PBST. After one hour, the blocking buffer wasdumped off of the plate and 100 μL/well of the phage was added andincubated at 25° C. for 1.5 hours with shaking. After binding, the platewas washed 10 times with PBST by manually filling the wells and dumpingoff the buffer between washes. Phage were eluted with 100 μL/well of 50mM HCl/500 mM KCl for 30 minutes at 25° C. with shaking. The elution wasneutralized with 100 μL/well of 1 M Tris, pH 7.5 and subsequentlypropagated in XL1-Blue (Agilent) E. coli with the addition of M13K07helper phage.

Amplified phage were used for additional rounds of selection againstlinear diubiquitin or K63-linked diubiquitin in plate-based sorting.Solution-based sorting was not possible because biotinylation of lineardiubiquitin interfered with 1E3 Fab binding. Stringency of the latersorts was increased in two ways: by increasing the number and durationof plate washes; and by decreasing the amount of phage used and theduration of phage binding. The second sort was done exactly as the firstsort, except that the amount of phage used was OD₂₆₈=0.5 and the numberof plate washes were increased to 21 with the last wash incubating at25° C. with shaking for 5 minutes. The third sort was done exactly asthe second sort, except that the duration of phage binding was reducedto one hour and the number of plate washes was increased to 30. For thelinear diubiquitin sort this was followed by four additional washes of15 minutes each with shaking at 25° C., with five quick washes inbetween each 15 minute wash. Then a one hour wash with shaking at 25° C.was performed followed by five quick washes. Enrichment was calculatedfor rounds two and three by comparing the number of phage recovered withlinear diubiquitin or K63-linked diubiquitin compared to an uncoatedwell. Strong enrichment was observed in rounds two and three for all sixlibraries sorted against linear diubiquitin with only modest enrichmentseen for K63-linked diubiquitin (see Table 4).

TABLE 4 Linear Linear K63 K63 diUb diUb diUb diUb Library Round 2 Round3 Round 2 Round 3 L1 10,000X 1,000X 0X 3-5X L2 10,000X 1,000X 0X 3-5X L310,000X 1,000X 0X 3-5X H1 10,000X 1,000X 0X 3-5X H2 10,000X 1,000X 0X 10X H3 10,000X 1,000X 0X  10X

After three rounds of sorting 64 individual clones were picked for eachof the six libraries from the linear diubiquitin second round sort, thelinear diubiquitin third round sort, and the K63-linked diubiquitinthird round sort and grown up in 96-well format in 1 mL of 2YT brothcontaining 50 μg/mL carbenicillin and 1×10¹⁰ phage/mL M13K07 helperphage. Supernatants from those cultures were used in high-throughputphage spot ELISAs for binding to 1 μg/mL coated linear diubiquitin(Boston Biochem), K63-linked diubiquitin (Boston Biochem), an anti-gDantibody (Genentech), or an uncoated well as previously described(Example 1B). The variable domains of these clones were also sequenced(see Table 5—linear diubiquitin and Table 6—K63-linked diubiquitin).Sequencing of H3 clones revealed that a T110A mutation was present insome clones outside of the region targeted for randomization in thelibrary design in addition to the intended mutation. This is likely dueto an oligonucleotide synthesis error or a mutagenesis error.

D) Single Spot Competition Phage ELISA

Single spot competition phage ELISAs were done to determine which cloneshad the biggest improvement in affinity for linear diubiquitin comparedto the parental 1E3 clone. The phage supernatants from the phage spotELISAs (Example 3C) were used. The competition ELISA was done asdescribed for the IC₅₀ ELISA (Example 1D) except phage supernatants wereused instead of purified phage and only a single concentration (25 nM)of soluble linear diubiquitin (Boston Biochem) was used. The competitionwas also done for each clone without addition of any soluble lineardiubiquitin to determine the phage binding signal in the absence of anycompeting antigen. The percent inhibition in binding in the presence of25 nM linear diubiquitin was calculated as [1−(OD₄₅₀ for 25 nMlinear/OD₄₅₀ for no linear)]×100%. The 1E3 parental clone showedvariable percent inhibition of binding ranging from 20% to 75%inhibition in the presence of 25 nM linear diubiquitin (see Table 5).This was due to variability in the OD₄₅₀ for binding linear diubiquitinin the absence of any competing soluble linear diubiquitin. Clonesshowing 60 percent inhibition or greater or those which were isolatedmany times in the linear diubiquitin sort were selected for furtheranalysis by phage IC₅₀ ELISA.

Table 5 below shows CDR L1, L2, L3, H1, H2 and H3 sequences from clonesisolated from the sorting of the 1E3 L1, L2, L3, H1, H2 and H3 NNKlibraries, respectively against linear diubiquitin. Also shown are theOD₄₅₀ signals from the competition spot ELISA in the absence andpresence of 25 nM soluble linear diubiquitin. The percent inhibition inbinding in the presence of 25 nM linear diubiquitin was calculated as[1−(OD₄₅₀ for 25 nM linear/OD₄₅₀ for no linear)]×100%. Table 6 belowshows CDR L1, L2, L3, H1, H2 and H3 sequences from clones isolated fromthe sorting of the 1E3 L1, L2, L3, H1, H2 and H3 NNK libraries,respectively, against K63-linked diubiquitin.

Table 5 discloses the CDR L1 sequences as SEQ ID NOS 1, 56, 199, 200,200, 57, 201, 202, 202, 203, 204, 204-207, 207, 208, 50, 50, 50, 50, 50,50, 209, 209, 54, 54, 54, 54, 210, 55, 55, 53, 211, 212, 212, 212-214,52, 52, 215, 215, 215, 216, 216, 217, 217, 217, 218, 1, 1, 1, 1, 1, 1,1, 1, 1, 1, 1, 1, 1, 1 and 1, respectively, in order of appearance.Table 5 discloses the CDR L2 sequences as SEQ ID NOS 2, 59, 59, 58, 58,58, 219, 60, 60, 60, 60, 60, 60, 60, 60, 60, 60, 220, 220, 220, 220,221, 221-223, 223-226, 226, 226, 62, 227, 228, 228, 229, 61, 61,230-233, 233-237, 237, 238, 238, 238, 239, 239, 239, 2, 2, 2, 2, 2, 2,2, 2, 2, 2, 2 and 2, respectively, in order of appearance. Table 5discloses the CDR L3 sequences as SEQ ID NOS 3, 6, 6, 240, 240, 240-242,66, 66, 66, 66, 243, 68, 244, 244-247, 247, 248, 248, 248, 70, 64, 64,64, 64, 249, 250, 250, 71, 71, 251, 72, 72, 72, 72, 72, 72, 72, 69, 65,65, 65, 252, 252, 252, 252, 252, 67, 67, 67, 67, 67, 67, 67, 67, 253, 3,3, 3, 3 and 254, respectively, in order of appearance. Table 5 disclosesthe CDR H1 sequences as SEQ ID NOS 255, 256, 73, 79, 79, 257, 257, 75,75, 75, 75, 77, 77, 77, 77, 77, 77, 77, 258, 258, 259, 81, 74, 74, 74,74, 74, 74, 74, 74, 74, 74, 74, 74, 74, 74, 78, 76, 76, 76, 76, 76, 76,76, 76, 76, 76, 80, 80, 80, 260, 260, 255, 255, 255, 255, 255, 255, 255,255, 255, 255, 255, 255 and 255, respectively, in order of appearance.Table 5 discloses the CDR H2 sequences as SEQ ID NOS 8, 17, 84, 261,261, 261, 261, 261-263, 85, 83, 83, 264, 82, 82, 82, 82, 82, 82, 82, 82,82, 82, 82, 86, 8, 8, 8, 8, 8, 8, 8, 8, 8, 8, 8, 8, 8, 8, 8, 8, 8, 8, 8,8, 8, 8, 8, 8, 8, 8, 8, 8, 8, 8, 8, 8, 8, 8, 8, 8 and 8, respectively,in order of appearance. Table 5 discloses the CDR H3 sequences as SEQ IDNOS 265-269, 269, 270-273, 273, 273, 273, 273, 273, 273, 273, 273, 273,273, 273, 273, 273, 273, 273, 273, 273, 273, 273, 273, 273, 273, 273,273, 273, 273, 273, 273, 273, 274, 274, 274, 274, 274-278, 278, 278,278-281, 281, 282, 282, 282, 282, 265, 265, 265, 265, 265, 265 and 265,respectively, in order of appearance.

Table 6 discloses the CDR L1 sequences as SEQ ID NOS 1, 283, 50, 50, 50,50, 50, 50, 50, 50, 50, 50, 50, 50, 50, 50, 50, 50, 50, 50, 50, 50, 50,284, 54, 285, 285-288, 55, 55, 55, 53, 289, 289, 290, 290, 290, 290,290, 290-294, 327, 295, 295, 295, 295-297, 297, 1, 1, 1, 1, 1, 1 and 1,respectively, in order of appearance. Table 6 discloses the CDR L2sequences as SEQ ID NOS 2, 298, 299, 60, 60, 300, 302-305, 305-307, 307,307-309, 309, 309, 310, 310-314, 314, 314, 314, 314, 314, 314, 314,314,314, 314, 315, 2, 2, 2, 2, 2, 2, 2, 2, 2, 2, 2, 2, 2, 2, 2, 2 and 2,respectively, in order of appearance. Table 6 discloses the CDR L3sequences as SEQ ID NOS 3, 6, 6, 6, 6, 6, 316-318, 318, 319, 66, 66,320, 321, 321, 321, 321-324, 324, 324, 325, 301, 326, 301, 326, 382,328, 328, 329, 64, 64, 330, 330, 331, 331, 331, 331, 331, 71, 71, 71,71, 71, 71, 332, 69, 333, 333, 67, 67, 67, 67, 67, 334, 334, 334, 335,335, 335, 3, 3, 3 and 3, respectively, in order of appearance. Table 6discloses the CDR H1 sequences as SEQ ID NOS 255, 336, 337, 337, 337,337, 337, 337, 337, 337, 337, 337, 337, 337, 337-340, 340-343, 343, 343,343, 343, 343, 344, 344, 344, 344, 344, 344, 81, 81, 81, 81, 81, 81, 74,74, 74, 74, 74, 74, 74, 74, 74, 74, 74, 74, 74, 74, 76, 76, 76, 76, 76,76, 76, 76, 76, 76 and 255, respectively, in order of appearance. Table6 discloses the CDR H2 sequences as SEQ ID NOS 8, 346, 347, 345, 348,349, 349, 349, 349, 349, 349, 349, 349, 349, 349, 349, 349, 349-351,351, 351, 351, 352, 352, 352, 353, 353, 353, 353, 353, 353, 353, 353,353, 353, 353, 353, 353, 353, 353, 353, 353, 353, 353, 353, 353, 353,353, 353, 353, 353, 353, 353, 353, 353, 353, 353, 353, 354, 8, 8, 8, 8,8 and 8, respectively, in order of appearance. Table 6 discloses the CDRH3 sequences as SEQ ID NOS 355-358, 358, 359, 359-362, 362, 362, 362,362, 362, 362, 362, 362, 362, 362, 362, 362, 362, 362, 362, 362, 362,362, 362, 362, 362, 362, 362, 362, 362, 362, 362, 362, 362, 362, 362,362, 362, 362, 362, 362, 362-367, 355, 355, 355, 355, 355, 355, 355,355, 355 and 355, respectively, in order of appearance. “O” is an ochrestop TAA and “q” is an amber stop TAG which can replaced by Gln (Q) forexample with the use of suppressor tRNA cell lines.

E) Phage IC₅₀ ELISA

Eight clones from the L1 library, six from the L2 library, nine from theL3 library, nine from the H1 library, five from the H2 library, andseven from the H3 library were tested in a phage IC₅₀ ELISA as describedin example 1D. Eight three-fold serial dilutions of linear diubiquitin(Boston Biochem) from 500 nM to 0.23 nM were used. In each experimentthe WT 1E3 clone was included for comparison. The IC₅₀ value for 1E3varies between experiments, however clones which show higher affinityfor linear diubiquitin than the parental clone can be identified. In anattempt to identify mutants with improved affinity for lineardiubiquitin with minimal K63 diubiquitin binding, clones that weretested in the IC₅₀ ELISA were narrowed down by taking into accountwhether they had improved IC₅₀ values compared to the parental 1E3,whether they were isolated in the K63-linked diubiquitin sort (example3C), as well as their signal for binding to K63-linked diubiquitin inthe phage spot ELISA (example 3C). Clones with improved IC₅₀ valuescompared to 1E3 that were isolated multiple times in the lineardiubiquitin sort but were not isolated in the K63-linked diubiquitinsort and demonstrated no K63-linked diubiquitin binding in the spotELISA (OD₄₅₀ of less than 0.1) were selected for further analysis (1F11,2A2, 2C11, 2H5, 3E4, 4C9, 4E4, and 4G7) (see Table 7). If a clone withan improved IC₅₀ that was isolated multiple times in the lineardiubiquitin sort, was isolated only once in the K63-linked diubiquitinsort, and demonstrated no K63-linked diubiquitin binding in the phagespot ELISA, then it was considered for further analysis (3F5) (see Table7). Clones 3A7 and 4C10 which were isolated multiple times in both thelinear diubiquitin and the K63-linked diubiquitin sorts were chosen asnegative controls. These 11 clones along with 1E3 were tested further ina phage specificity ELISA. Table 7 below shows the CDR L1, L2, L3, H1,H2 and H3 sequences of clones from the sorting of the 1E3 L1, L2, L3,H1, H2 and H3 NNK libraries, respectively, against linear diubiquitinthat were further characterized by phage IC₅₀ ELISA. The IC₅₀ and thefold improvement over the parental 1E3 IC₅₀ is given. Binding toK63-linked diubiquitin is defined as having an OD₄₅₀ of greater than 0.1in the phage spot ELISA. Also shown is the percent inhibition in bindingin the presence of 25 nM soluble linear diubiquitin in the phage spotcompetition ELISA. The number of times each clone was isolated in thelibrary sorts against linear diubiquitin or K63-linked diubiquitin isindicated.

Table 7 discloses the CDR L1 sequences as SEQ ID NOS 1, 1 and 50-57,respectively, in order of appearance. Table 7 discloses the CDR L2sequences as SEQ ID NOS 2 and 58-63, respectively, in order ofappearance. Table 7 discloses the CDR L3 sequences as SEQ ID NOS 3, 3,64, 65, 3 and 66-72, respectively, in order of appearance. Table 7discloses the CDR H1 sequences as SEQ ID NOS 255 and 73-81,respectively, in order of appearance. Table 7 discloses the CDR H2sequences as SEQ ID NOS 8 and 82-86, respectively, in order ofappearance. Table 7 discloses the CDR H3 sequences as SEQ ID NOS368-372, 368 and 373-375, respectively, in order of appearance.

F) Phage Specificity ELISA

1F11, 2A2, 2C11, 2H5, 3E4, 4C9, 4E4, 4G7, 3F5, 3A7, and 4C10 along withthe parental clone 1E3 were tested in a phage specificity ELISA againstmonoubiquitin (Boston Biochem), linear diubiquitin (Boston Biochem), K11-linked diubiquitin (Genentech), K48-linked diubiquitin (BostonBiochem), K63-linked diubiquitin (Boston Biochem), an anti-gD antibody(Genentech), or an uncoated well. The panel of ubiquitin proteins wasimmobilized on 96-well Maxisorb immunoplates (NUNC) (see FIGS. 7A and7B). Plates were coated at 4° C. overnight with 1 μg/mL of each proteinin 50 mM sodium carbonate buffer, pH 9.6. The coated plates were blockedwith 200 μL/well of 2.5% milk in PBST for one hour at 25° C. withshaking. Six two-fold serial dilutions of phage from OD₂₆₈=1.0 toOD₂₆₈=0.03 were made in 2.5% milk in PBST. After one hour, the blockingbuffer was dumped off of the plate and 100 μL of the phage serialdilutions was added. Plates were incubated at 25° C. for one hour withshaking. The plate was then washed six times with PBST using a platewasher. A 1:5,000 dilution of an anti-M13 horseradish peroxidase(HRP)-conjugated secondary antibody (GE Healthcare) in PBST was used fordetection of phage binding. 100 μL/well of the secondary dilution wasadded and the plate was incubated at 25° C. for 1.25 hours with shaking.The plate was then washed six times with PBST using a plate washer andtwice manually with PBS. Bound secondary antibody was detected using aTMB substrate (KPL) followed by quenching with an equal volume of 1 Mphosphoric acid. The absorbance was read at 450 nm.

Clones 3A7 and 4C10 which were isolated multiple times in both thelinear diubiquitin and the K63-linked diubiquitin sorts were used asnegative controls. Both demonstrated significant K63-linked binding at aphage OD₂₆₈=1.0 (OD₄₅₀=0.44 and OD₄₅₀=0.5 for 3A7 and 4C10,respectively) (see FIGS. 7A and 7B). 2A2 which was isolated twice in theK63-linked diubiquitin sort showed intermediate levels of K63-linkedbinding (OD₄₅₀=0.2 at a phage OD₂₆₈=1.0). All other clones demonstratednegligible K63-linked diubiquitin binding (OD₄₅₀<0.16 at a phageOD₂₆₈=1.0).

G) Cloning Light Chain/Heavy Chain Double Mutants

Clones which demonstrated improved IC₅₀s over the parental 1E3 (example3E) and showed negligible K63-linked diubiquitin binding in the phagespecificity ELISA (example 3F) were chosen for further consideration.These included light chain mutants 1F11, 2C11, and 2H5 and heavy chainmutants 3E4, 3F5, and 4G7. To see whether there was an additive effectin affinity improvement double mutants were constructed combiningdifferent combinations of light and heavy chain mutations. Light chainvariable domains were removed by digesting the phagemids with EcoRV andKpnI and were then cloned into the various heavy chain mutant phagemidsusing the same sites.

H) Double Mutant Phage IC₅₀ ELISA

Double mutants 1F11/3E4, 1F11/3F5, 1F11/4G7, 2C11/3E4, 2C11/3F5,2C11/4G7, 2H5/3E4, 2H5/3F5, and 2H5/4G7 were compared to theirrespective single mutants and parental 1E3 in a phage IC₅₀ ELISA aspreviously described (examples 1D and 3E). Double mutants 1F11/3F5 and2H5/3F5 demonstrated an additive improvement in affinity over theirindividual single mutants (see Table 8). 2C11/3F5 was border lineadditive. The single mutant 3F5 in this particular assay gave a lowerIC₅₀ (9 nM) than in the other two experiments (12 or 13 nM) which couldmake 2C11/3F5 (IC₅₀ of 10 nM) appear not to be additive. Therefore2C11/3F5 was also chosen for further consideration.

TABLE 8 IC50 (nM) CDR mutation WT 29 1F11 14 L2 S52K 3E4 44 H1 I34M 3F512 H2 S56Q 4G7 23 H3 Y102G 1F11/3E4 12 L2/H1 S52K 134M 1F11/3F5  5 L2/H2S52K S56Q 1F11/4G7 13 L2/H3 S52K Y102G WT 24 2C11 20 L3 T94Q 3E4 33 H1I34M 3F5  9 H2 S56Q 4G7 16 H3 Y102G 2C11/3E4 24 L3/H1 T94Q 134M 2C11/3F510 L3/H2 T94Q S56Q 2C11/4G7 31 L3/H3 T94Q Y102G WT 25 2H5 17 L3 T94N 3E426 H1 I34M 3F5 13 H2 S56Q 4G7 19 H3 Y102G 2H5/3E4 26 L3/H1 T94N 134M2H5/3F5  8 L3/H2 T94N S56Q 2H5/4G7 23 L3/H3 T94N Y102G

I) Double Mutant Phage Specificity ELISA

Double mutants 1F11/3F5, 2C11/3F5, and 2H5/3F5 were compared to theirrespective single mutants and parental 1E3 in a phage specificity ELISAas in example 3F. All double mutants showed negligible K63-linkeddiubiquitin binding (OD₄₅₀<0.1 at phage OD₂₆₈=1.0) (see FIGS. 8A and8B).

J) Double Mutant Fab Production

The parental clone (1E3), single mutants (1F11, 2C11, 2H5, 3F5) anddouble mutants (1F11/3F5, 2C11/3F5, 2H5/3F5) were cloned as Fabexpression constructs by inserting a TAA stop codon into the phagemidsat the end of the CH1 domain as described in example 1E. These Fabs wereexpressed in E. coli and purified as described in example 1E.

K) Conversion to IgG Format

The parental clone (1E3), single mutants (1F11, 2C11, 2H5, 3F5) anddouble mutants (1F11/3F5, 2C11/3F5, 2H5/3F5) were also expressed inHEK293 cells as human immunoglobulins (IgGs). Expression constructs weregenerated by cloning the Fab variable domains into pRK mammalianexpression constructs encoding the heavy and light chains of human kappaIgG1 (Gorman et al., DNA Prot. Eng. Tech. 2:3-10 (1990)). IgGs werepurified by affinity chromatography on protein A-sepharose columns bystandard methodologies as described for the Fab purification in Example1E.

L) Double Mutant Biacore

The affinity of the double mutant Fabs (from Example 3J) was analyzed bysurface plasmon resonance (SPR) using a BIACORE™ 3000 (GE Healthcare)and direct binding as described in example 1G. Approximately 150resonance units (RUs) of linear diubiquitin (Boston Biochem), K48-linkeddiubiquitin (Boston Biochem), and K63-linked diubiquitin (BostonBiochem) were immobilized on flow cell two, flow cell three, and flowcell four, respectively, of a CM5 chip using the amine coupling protocolsupplied by the manufacturer. Flow cell one was activated andethanolamine blocked without immobilizing protein, to be used forreference subtraction. Even with 10 mM glycine, pH 1.7 the chip surfacecould not be regenerated back to baseline and an increase in RUs wasseen suggesting that the chip surface was altered.

An alternative approach was tested using an IgG capture method with theIgGs from example 3K on a BIACORE™ 3000 (GE Healthcare). Approximately8,000 resonance units (RUs) of an anti-human Fc capture antibody (GEHealthcare) were immobilized on flow cells one and two of a CM5 chipusing the amine coupling protocol supplied by the manufacturer. 60 μL of1 μg/mL IgG in 10 mM Hepes, pH 7.2, 150 mM NaCl, and 0.01% Tween 20(HBST) was injected at a flow rate of 30 μL/minute over flow cell two,resulting in capture of approximately 750 RUs of IgG. Flow cell one hadonly the capture antibody on it to serve as a reference subtraction.Two-fold serial dilutions (3.9-500 nM) of linear diubiquitin (BostonBiochem) or K63-linked diubiquitin (Boston Biochem) in HBST wereinjected (60 μL total at a flow rate of 30 μL/minute) over flow cellsone and two. The signal for each flow cell was recorded and thereference signal was subtracted. Following a dissociation period of fourminutes, the chip surface was regenerated with one injection of 15 μL of3M MgCl₂ at a flow rate of 30 μL/minute. Much like the Fab captureBiacore experiment (see example 1G), data were difficult to fit to anybinding model because the diubiquitin did not fully dissociate from thechip. In addition the association rates were very fast and binding didnot reach a plateau even at the highest concentration of diubiquitinused. Therefore it is difficult to estimate a KD for these IgGs.

M) Western Blots of Purified Diubiquitin with Double Mutants

To rank the affinity and specificity of the double mutants the IgGsdescribed in example 3K were tested in a western blot for binding tolinear diubiquitin (Boston Biochem) and K63-linked diubiquitin (BostonBiochem). 1 μg of K63-linked diubiquitin and five three-fold serialdilutions of linear diubiquitin (1000, 333, 111, 37, 12 ng) in 1×LDSbuffer (Invitrogen) with reducing agent was heated at 70° C. for tenminutes and run on 4-12% NuPAGE Bis Tris 1.0 mm gels in MES buffer(Invitrogen). Gels were transferred at 30 V constant for 1 hour by wettransfer in 10% methanol and 1×NuPAGE transfer buffer (Invitrogen) to0.2 μm nitrocellulose (Invitrogen). Non-specific binding sites on themembranes were blocked by incubation in 5% milk in PBST for 1 hour at25° C. with shaking. The membranes were then incubated in 1 μg/mL of1E3, 1F11, 2C11, 2H5, 3F5, 1F11/3F5, 2C11/3F5, or 2H5/3F5 IgG in 5% milkin PBST for one hour at 25° C. with shaking. The membranes were washedthree times in PBST with shaking. The IgGs were detected by incubatingthe membrane in a 1:10,000 dilution of a goat anti-human Fcfragment-specific IR Dye 800CW-conjugated secondary antibody (RocklandImmunochemicals) in 5% milk in PBST for 30 minutes at 25° C. withshaking. The membranes were then washed three times in PBST followed byone wash in PBS. The secondary antibody was detected and quantifiedusing the LI-COR Odyssey infrared imaging system (LI-COR Biosciences).

Single mutants 1F11 and 3F5 were considerably more sensitive than theparental 1E3, whereas single mutants 2C11 and 2H5 were only slightlyimproved (see FIG. 10 ). The double mutant 1F11/3F5 had an additiveimprovement in sensitivity over the respective single mutants, whereas2C11/3F5 and 2H5/3F5 were not any better than 3F5 alone.

N) Cloning Triple Mutants

To see whether a further improvement in affinity could be achieved bycombining three mutations, the 1F11/2C11/3F5 and 1F11/2H5/3F5 triplemutants were generated. Mutagenic oligonucleotides 5′-1F11 S52K(CCGAAGCTTCTGATTTACTCGGCAAAGTTCCTCTA CTCTGGAGTCCC) (SEQ ID NO:187) and3′-1F11 S52K (GGGACTCCAGAGTAG AGGAACTTTGCCGAGTAAATCAGAAGCTTCGG) (SEQ IDNO:188) were combined with either the 2C11 or 2H5 pRK light chainconstructs from example 3K and the QuikChange® Lightning Site-DirectedMutagenesis kit (Agilent) was used to generate the double mutant lightchains. Triple mutants were then generated by combining the doublemutant light chain pRK constructs with the 3F5 heavy chain pRK constructand IgGs were expressed in HEK293 cells and purified as described inexample 3K.

O) Western Blots of Purified Diubiquitin with Triple Mutants

To rank the affinity and specificity of the triple mutants 1F11/2C11/3F5and 1F11/2H5/3F5 the IgGs described in example 3N were tested in awestern blot for binding to linear diubiquitin (Boston Biochem) andK63-linked diubiquitin (Boston Biochem) as described in example 3M.Neither 1F11/2C11/3F5 nor 1F11/2H5/3F5 was more sensitive than thedouble mutant 1F11/3F5 (see FIG. 11 ).

P) Cloning Additional Triple Mutants

One H3 mutant, 4E4 that demonstrated an improved IC₅₀ value over theparental 1E3 in example 3E was not originally considered when makingdouble mutants because it had a small amount of K63-linked diubiquitinbinding in the specificity ELISA (OD₄₅₀=0.16 at a phage OD₂₆₈=1.0, seeexample 3F). Since none of the IgG mutants tested so far demonstratedany K63-linked diubiquitin binding in the western blots, the 4E4 clonewas analyzed further. 4E4 was an H3 clone that actually contained twomutations, Y102L immediately adjacent to CDR H3 and T 110A in framework4. To determine whether the unintentional T 110A mutation had any affecton affinity, both the Y102L single mutant and the Y102L Ti 10A doublemutant heavy chain was made in the context of the 1E3 parental heavychain or the mutant 3F5 heavy chain. To insert the Y102L mutationmutagenic oligonucleotides 5′-Y102L(CGGTGGGTTATGGACCTGTGGGGTCAAGGAACCCTGGTC ACCGTCTCCTCGGCCTCC) (SEQ IDNO:189) and 3′-Y102L (GGAGGCCGAGGAGACGGTGACCAGGGTTCCTTGACCCCACAGGTCCATAACCCACCG) (SEQ ID NO:190) werecombined with either the 1E3 or 3F5 IgG heavy chain pRK expressionconstruct and the mutants were synthesized using the QuikChange®Lightning Site-Directed Mutagenesis kit (Agilent). To insert the Y102LT110A mutations mutagenic oligonucleotides 5′-Y102L T110A(CGGTGGGTTATGGACCTGTGGGGTCAAGGAACCCTGGTCGCGGTCTCCTCGGCCTCC) (SEQ IDNO:191) and 3′-Y102L T110A (GGAGGCCGAGGAGACCGCGACCAGGGTTCCTTGACCCCACAGGTCCATAACCCACCG) (SEQ ID NO:192) were combined witheither the 1E3 or 3F5 IgG heavy chain pRK expression construct and themutants were synthesized using the QuikChange® Lightning Site-DirectedMutagenesis kit (Agilent). The resulting heavy chain IgG pRK constructswere combined with either the parental 1E3 or the mutant 1F11 lightchain IgG pRK constructs and the resulting IgGs were expressed in HEK293cells and purified as described in example 3K.

These mutants (Y012L vs. Y102L Ti 10A, 3F5/Y102L vs. 3F5/Y102L T110A,1F11/Y102L vs. 1F11/Y102L T110A, and 1F11/3F5/Y102L vs. 1F11/3F5/Y102LT110A) were then compared side by side in a western blot for bindinglinear or K63-linked diubiquitin as described in example 3M. Generally,having the T110A mutation in combination with Y102L did notsignificantly improve sensitivity compared to Y102L alone (see FIG. 12). Also from the phage IC₅₀s it is known that Ti 10A alone (clone 4E1)does not improve affinity compared to the parental 1E3 (see Table 7).Therefore the Y102L mutation alone was considered for further analysis.

The triple mutant 1F11/3F5/Y102L was then compared to the parental 1E3,each of the single mutants (1F11, 3F5, Y102L), as well as the doublemutants (1F11/3F5, 1F11/Y102L, 3F5/Y102L) side by side in a western blotfor binding linear or K63-linked diubiquitin as described in example 3M.The triple mutant was more sensitive than the parental clone 1E3, all ofthe single mutants, and all of the double mutants for binding lineardiubiquitin (see FIG. 13 ). In addition it showed no binding toK63-linked diubiquitin indicating that the specificity is maintained.

Example 4—Characterization of the Affinity Matured Anti-LinearPolyubiquitin Antibody

A) IgG Western Blot of Purified Diubiquitin Chains

The 1E3 parental and 1F11/3F5/Y102L IgGs were tested for their abilityto detect pure diubiquitin chains in a western blot. Seven two-foldserial dilutions of linear diubiquitin (Boston Biochem) from 1 μg to 16ng and 1 μg each of monoubiquitin (Boston Biochem), K11-linkeddiubiquitin (Genentech), K48-linked diubiquitin (Boston Biochem), andK63-linked diubiquitin (Boston Biochem) in 1×LDS buffer (Invitrogen)with reducing agent (Invitrogen) were heated at 70° C. for ten minutesand run on 4-12% Bis Tris NuPAGE 1.0 mm gels (Invitrogen) in MES buffer(Invitrogen) in triplicate. One gel was stained by SimplyBlue Coomassiestain (Invitrogen) to detect all proteins. For comparison to get an ideaof affinity, a second western blot was done with the anti-K63 antibody,Apu3. A8 which has a known KD of 8.7 nM for K63-linked diubiquitin (seeNewton, K. et al. (2008) Cell 134:668-678). For this western, seventwo-fold serial dilutions of K63-linked diubiquitin (Boston Biochem)from 1 μg to 16 ng and 1 μg each of monoubiquitin (Boston Biochem),linear diubiquitin (Boston Biochem), K11-linked diubiquitin (Genentech),and K48-linked diubiquitin (Boston Biochem) were run on the gel asdescribed above. The three gels were transferred individually at 30 Vconstant for one hour by wet transfer in 10% methanol and 1×NuPAGEtransfer buffer (Invitrogen) to 0.2 μm nitrocellulose (Invitrogen).Non-specific binding sites on the membranes were blocked by incubationin 5% milk in PBST for one hour at 25° C. with shaking. The membraneswere then incubated in 1 μg/mL of 1E3, 1F11/3F5/Y102L, or Apu3. A8 in 5%milk in PBST for one hour at 25° C. with shaking. The membranes werewashed three times in PBST with shaking. The IgGs were detected byincubating the membranes in a 1:10000 dilution of a goat anti-humanFcγ-specific HRP-conjugated F(ab′)2 secondary antibody (JacksonImmunoresearch) in 5% milk in PBST for one hour at 25° C. with shaking.The membranes were then washed three times in PBST followed by one washin PBS. The secondary antibody was detected using Super Signal West Picochemiluminescent substrate (Thermo Scientific) followed by exposure ofthe blots to film.

The 1E3 limit of detection was ˜250 ng of linear diubiquitin (see FIG.14 ). In contrast, 1F11/3F5/Y102L was much more sensitive and coulddetect as little as 31 ng of linear diubiquitin. In addition1F11/3F5/Y102L was highly specific, demonstrating no binding to any ofthe other forms of ubiquitin tested. For comparison the limit ofdetection of Apu3. A8, which has a K_(D) of 8.7 nM, was ˜62 ng. TheK_(D) of 1F11/3F5/Y102L for linear diubiquitin is therefore likely inthe low nM range.

B) IgG Western Blot of Purified Polyubiquitin Chains

The linear-specific antibodies were generated against a lineardiubiquitin antigen so they presumably recognize either the linkageitself or the surrounding surface residues on the proximal and distalubiquitins that are placed in close proximity due to the conformation ofdiubiquitin which results from the linear linkage. Since diubiquitin isthe smallest recognition unit of antigen and linear polyubiquitin is apolymeric chain with diubiquitin as the repeating “monomer” unit, theantibodies should also bind the polyubiquitin form. To examine this, the1F11/3F5/Y102L IgG was tested for its ability to detect purepolyubiquitin chains in a western blot. 1 μg each of monoubiquitin(Boston Biochem), linear polyubiquitin 2-7 (Enzo Lifesciences),K11-linked polyubiquitin (Genentech), K48-linked polyubiquitin 2-7(Boston Biochem), and K63-linked polyubiquitin 2-7 (Boston Biochem) in1×LDS buffer (Invitrogen) with reducing agent (Invitrogen) was heated at70° C. for ten minutes and run on 4-12% Bis Tris NuPAGE 1.0 mm gels(Invitrogen) in MES buffer (Invitrogen) in triplicate. One gel wasstained by SimplyBlue Coomassie stain (Invitrogen) to detect allproteins. The other two gels were transferred separately at 30 Vconstant for one hour by wet transfer in 10% methanol and 1×NuPAGEtransfer buffer (Invitrogen) to 0.2 μm nitrocellulose (Invitrogen).Non-specific binding sites on the membranes were blocked by incubationin 5% milk in PBST for one hour at 25° C. with shaking. The membraneswere then incubated in 1 μg/mL of 1F11/3F5/Y102L IgG or a 1:200 dilutionof a mouse pan-ubiquitin antibody, P4D1 (non-linkage specific, SantaCruz Biotechnology) in 5% milk in PBST for one hour at 25° C. withshaking. The membranes were washed three times in PBST with shaking. The1F11/3F5/Y102L IgG was detected by incubating the membrane in a 1:10000dilution of a goat anti-human Fcγ-specific HRP-conjugated F(ab′)2secondary antibody (Jackson Immunoresearch) in 5% milk in PBST for onehour at 25° C. with shaking. The P4D1 IgG was detected by incubating themembrane in a 1:10,000 dilution of a goat anti-mouse Fcγ-specificHRP-conjugated F(ab′)2 secondary antibody (Jackson Immunoresearch) in 5%milk in PBST for one hour at 25° C. with shaking. The membranes werethen washed three times in PBST followed by one wash in PBS. Thesecondary antibodies were detected using Super Signal West Picochemiluminescent substrate (Thermo Scientific) followed by exposure ofthe blots to film. Whereas the control pan-ubiquitin antibody, P4D1recognizes monoubiquitin, linear polyubiquitin 2-7 (albeit poorly),K11-linked polyubiquitin, K48-linked polyubiquitin 2-7, and K63-linkedpolyubiquitin 2-7, the 1F11/3F5/Y102L IgG recognizes only linearpolyubiquitin (see FIG. 15 ). Thus, just as with linear diubiquitin, the1F11/3F5/Y102L antibody can detect polyubiquitin chains containing thelinear linkage, but does not recognize polyubiquitin chains of otherlinkages.

C) IgG Western Blot of TNFα-Treated Cell Lysates

HeLa S3 cells were grown in suspension culture in 50:50 F-12: Dulbecco'sModified Eagle Media (DMEM) supplemented with 10% fetal bovine serum(FBS), 2 mM L-glutamine, 1% glycine/hypoxanthine/thymidine (GHT)solution, and 1% penicillin/streptomycin. The day before the experimentthe cells were split 1:2. The cells were grown overnight until reachinga density of 0.38×106 cells/mL (98% viable). The cells were divided intothree flasks of 1.5 L of cells each. Flask 1 was pretreated with 5.8 μMMG132 (Cayman Chemicals) for 10 minutes. Flask 2 and 3 received nopretreatment. At time zero flasks 2 and 3 also were treated with 5.8 μMMG132. In addition at time zero, flasks 1 and 3 were treated with 100ng/mL TNFα (Shenandoah Biotechnology) and flask 2 was treated with 500ng/mL TNFα. At time zero, five minutes, and 20 minutes 444 mL of cellswere removed from each flask, spun down at 800 rpm for five minutes at4° C., and the supernatants were aspirated. Cells were immediatelywashed with 40 mL of cold PBS, pelleted at 800 rpm for five minutes at4° C., and the supernatants were aspirated. Each pellet was lysed in 13mL of cold lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1%Triton X-100, 10 mM N-ethylmaleimide (NEM), 25 μM MG132, 50 mM NaF,Complete protease inhibitor cocktail tablets (Roche), and PhosSTOPphosphatase inhibitor cocktail tablets (Roche)) for 10 minutes at 4° C.with rocking. Debris was pelleted by spinning at 10,000×g for fiveminutes at 4° C. Lysates were precleared with 133 μL of Protein ADynabeads (Invitrogen) for 1 hour at 4° C. with rocking. Beads werepelleted by spinning at 2000 rpm for five minutes. The supernatant wasremoved and stored at −80° C.

Linear polyubiquitin chains have been suggested to play a signaling rolein the NFκB pathway. Therefore the above lysates were probed with1F11/3F5/Y102L in a western blot. Thirteen μL of each lysate in 1×LDSsample buffer (Invitrogen) with reducing agent (Invitrogen) was heatedat 70° C. for ten minutes and run on 4-12% Bis Tris NuPAGE 1.0 mm gels(Invitrogen) in MES buffer (Invitrogen) in duplicate. As specificitycontrols 250 ng of purified linear (Enzo Lifesciences) and K63-linkedpolyubiquitin chains (Boston Biochem) were run on the gel. The gels weretransferred individually at 30 V constant for one hour by wet transferin 10% methanol and 1×NuPAGE transfer buffer (Invitrogen) to 0.45 μmnitrocellulose (Invitrogen). Non-specific binding sites on the membraneswere blocked by incubation in 5% milk in PBST for one hour at 25° C.with shaking. The membranes were then incubated in 1 μg/mL of1F11/3F5/Y102L or Apu3. A8 anti-K63 in 5% milk in PBST for one hour at25° C. with shaking. The membranes were washed three times in PBST withshaking. The IgGs were detected by incubating the membranes in a1:10,000 dilution of a goat anti-human Fcγ-specific HRP-conjugatedF(ab′)2 secondary antibody (Jackson Immunoresearch) in 5% milk in PBSTfor one hour at 25° C. with shaking. The membranes were then washedthree times in PBST followed by one wash in PBS. The secondary antibodywas detected using Super Signal West Pico chemiluminescent substrate(Thermo Scientific) followed by exposure of the blots to film. Anadditional western blot was done to assess activation of the NFκBpathway by probing for IκBα levels. Upon TNFα signaling this leads toubiquitination and degradation of the inhibitor of NFκB, IκBα. Five μLof the above lysates in 1×LDS sample buffer (Invitrogen) with reducingagent (Invitrogen) was heated at 70° C. for ten minutes and run on a4-12% Bis Tris NuPAGE 1.0 mm gel (Invitrogen) in MES buffer (Invitrogen)in duplicate. The gel was transferred at 30 V constant for two hours bywet transfer in 20% methanol and 1×NuPAGE transfer buffer (Invitrogen)to Invitrolon PVDF (Invitrogen). The membrane was blocked in 5% milk inPBST for one hour at 25° C. with shaking and then probed with 1:1000dilutions of an anti-IκBa (Cell Signaling) and an anti-p-tubulin (CellSignaling) antibody as a loading control at 4° C. overnight withshaking. The following day the blots were washed three times in PBSTwith shaking. The IgGs were detected by incubating the membranes in a1:10,000 dilution of a goat anti-rabbit Fcγ-specific HRP-conjugatedF(ab′)2 secondary antibody (Jackson Immunoresearch) in 5% milk in PBSTfor one hour at 25° C. with shaking. The membranes were then washedthree times in PBST followed by one wash in PBS. The secondary antibodywas detected using Super Signal West Pico chemiluminescent substrate(Thermo Scientific) followed by exposure of the blots to film.

In cells which were stimulated with 100 ng/mL TNFα and no pretreatmentwith MG132, the amount of linear polyubiquitin chains increases fromtime zero to five minutes to 20 minutes (see FIG. 16A). In contrast,K63-linked polyubiquitin chains which were much more abundant at allthree time points showed an increase from zero to five minutes and thena decrease back down to the starting levels at 20 minutes. In cellswhich were stimulated with 500 ng/mL TNFα and no pretreatment withMG132, the linear polyubiquitin chains demonstrated the same pattern ofincreasing from zero to 20 minutes, however the abundance of the linearchains at each time point was increased compared to the cells treatedwith 100 ng/mL TNFα. In contrast, the K63-linked chains demonstrated thesame pattern and abundance as compared to the cells treated with 100ng/mL TNFα. In cells which were pretreated for 10 minutes with 5.8 μMMG132 and then 100 ng/mL TNFα, the levels of both linear and K63-linkedchains remained fairly constant over the three time points. The blot forIκBa demonstrates that under all three experimental conditions the NFκBpathway is activated as evidenced by degradation of IκBa upon TNFαtreatment over time, however the extent of activation in greater in theabsence of MG132 pretreatment (see FIG. 16B). This demonstrates that1F11/3F5/Y102L can recognize endogenous linear polyubiquitin chains andsuggests that these chains are up regulated in HeLa S3 cells upon TNFαstimulation.

D) Immunoprecipitation of Linear Polyubiquitin Chains

The 1F11/3F5/Y102L IgG was tested to see whether it is capable ofimmunoprecipitating linear polyubiquitin chains. As a positive controlthe Apu3. A8 anti-K63 antibody was also used to monitorimmunoprecipitation of K63-linked chains. As a negative control anunrelated human kappa IgG1 antibody was used as an isotype control.Three immunoprecipitation (IP) conditions were tested. In reaction 1which contained all chains, 2 μg each of monoubiquitin (Boston Biochem),linear polyubiquitin 2-7 (Enzo Lifesciences), K11-linked polyubiquitin(Genentech), K48-linked polyubiquitin 2-7 (Boston Biochem), andK63-linked polyubiquitin 2-7 (Boston Biochem) were mixed. In reaction 2which lacked linear chains, 2 μg each of monoubiquitin, K11-linkedpolyubiquitin, K48-linked polyubiquitin 2-7, and K63-linkedpolyubiquitin 2-7 were mixed. Reaction 3 consisted of 2 μg of linearpolyubiquitin 2-7 chains alone. Each reaction was diluted in 500 μL of 4M urea IP buffer (4 M urea, 20 mM Tris, pH 7.5, 135 mM NaCl, 1% TritonX-100, 10% glycerol, 1 mM EDTA, 1.5 mM MgCl₂). The reactions wereprecleared with 50 μL of Protein A Dynabeads (Invitrogen) for threehours at 25° C. with rotation. The beads were then captured on amagnetic stand and the supernatants were transferred to new tubes.Twenty μg of 1F11/3F5/Y102L anti-linear, Apu3. A8 anti-K63, or anisotype control IgG was added to each IP reaction and incubatedovernight at 25° C. with rotation. The following day 100 μL of Protein ADynabeads were added to each reaction and the IgGs were captured for 15minutes at 25° C. with rotation. The beads were then washed three timeswith 1 mL each of 4M urea IP buffer followed by two washes with 1 mLeach of PBS. During the final wash the beads were transferred to newtubes to avoid eluting any proteins bound to the tube walls. The beadswere resuspended in 30 μL of 1×LDS sample buffer (Invitrogen) withreducing agent (Invitrogen) and heated at 70° C. for 10 minutes to elutethe immunoprecipitated proteins. The beads were then captured on amagnetic stand and the supernatant was split in half and loaded induplicate onto two 4-12% Bis Tris NuPAGE 1.0 mm gels (Invitrogen). Aspositive and negative controls 1 μg each of purified linearpolyubiquitin 2-7 (Enzo Lifesciences) and K63-linked polyubiquitin 2-7(Boston Biochem) were also run on the gels. The gels were run in MESbuffer (Invitrogen) and were then transferred individually at 30 Vconstant for one hour by wet transfer in 10% methanol and 1×NuPAGEtransfer buffer (Invitrogen) to 0.2 μm nitrocellulose (Invitrogen).Non-specific binding sites on the membranes were blocked by incubationin 5% milk in PBST for one hour at 25° C. with shaking. The membraneswere then incubated in 1 μg/mL of 1F11/3F5/Y102L or Apu3. A8 anti-K63 in5% milk in PBST for 1.5 hours at 25° C. with shaking. The membranes werewashed three times in PBST with shaking. The IgGs were detected byincubating the membranes in a 1:10,000 dilution of a goat anti-humanFc-specific HRP-conjugated F(ab′)2 secondary antibody (JacksonImmunoresearch) in 5% milk in PBST for one hour at 25° C. with shaking.The membranes were then washed three times in PBST followed by one washin PBS. The secondary antibody was detected using Super Signal West Picochemiluminescent substrate (Thermo Scientific) followed by exposure ofthe blots to film. 1F11/3F5/Y102L is able to immunoprecipitate linearpolyubiquitin chains in 4M urea however it is not specific under theseconditions as it is also able to pull down K63-linked chains (see FIG.17 ).

To determine whether different concentrations of urea could help improvespecificity, immunoprecipitations were carried out under differentbuffer conditions. Two μg each of linear polyubiquitin 2-7 andK63-linked polyubiquitin 2-7 were mixed and diluted with 500 μL of IPbuffer (20 mM Tris, pH 7.5, 135 mM NaCl, 1% Triton X-100, 10% glycerol,1 mM EDTA, 1.5 mM MgCl₂) containing 0, 2, 4, or 6 M urea. An additionalIP was done using 500 μL of PBST. The reactions were precleared with 50μL of Protein A Dynabeads (Invitrogen) for 30 minutes at 25° C. withrotation. The beads were then captured on a magnetic stand and thesupernatants were transferred to new tubes. Twenty μg of 1F11/3F5/Y102Lanti-linear or Apu3. A8 anti-K63 IgG was added to each IP reaction andincubated at 25° C. with rotation for one hour. Next 100 μL of Protein ADynabeads were added to each reaction and the IgGs were captured for 15minutes at 25° C. with rotation. The beads were then washed three timeswith 1 mL each of the corresponding buffer used in the IP (0, 2, 4, or 6M urea IP buffer or PBST) followed by two washes with 1 mL each of PBS.During the final wash the beads were transferred to new tubes to avoideluting any proteins bound to the tube walls. The beads were resuspendedin 20 μL of 1×LDS sample buffer (Invitrogen) with reducing agent(Invitrogen) and heated at 70° C. for 10 minutes to elute theimmunoprecipitated proteins. The beads were then captured on a magneticstand and the supernatant was split in half and loaded in duplicate ontotwo 4-12% Bis Tris NuPAGE 1.0 mm gels (Invitrogen). As positive andnegative controls 1 μg each of purified linear polyubiquitin 2-7 andK63-linked polyubiquitin 2-7 were also run on the gels. The gels wererun in MES buffer (Invitrogen) and were then transferred individually at30 V constant for one hour by wet transfer in 10% methanol and 1×NuPAGEtransfer buffer (Invitrogen) to 0.2 μm nitrocellulose (Invitrogen).Non-specific binding sites on the membranes were blocked by incubationin 5% milk in PBST for one hour at 25° C. with shaking. The membraneswere then incubated in 1 μg/mL of 1F11/3F5/Y102L or Apu3. A8 anti-K63 in5% milk in PBST for one hour at 25° C. with shaking. The membranes werewashed three times in PBST with shaking. The IgGs were detected byincubating the membranes in a 1:10,000 dilution of a goat anti-humanFc-specific HRP-conjugated F(ab′)2 secondary antibody (JacksonImmunoresearch) in 5% milk in PBST for one hour at 25° C. with shaking.The membranes were then washed three times in PBST followed by one washin PBS. The secondary antibody was detected using Super Signal West Picochemiluminescent substrate (Thermo Scientific) followed by exposure ofthe blots to film. As the concentration of urea is increased in the IPbuffer, the 1F11/3F5/Y102L IP becomes more specific (see FIG. 18A). At 6M urea very little K63-linked polyubiquitin is pulled down by the1F11/3F5/Y102L IgG and yet it is still able to pull down a significantamount of linear polyubiquitin.

To see whether even higher concentrations of urea could further improvespecificity the IPs were repeated using 6, 7, or 8 M urea IP buffer. Twoμg each of linear polyubiquitin 2-7 and K63-linked polyubiquitin 2-7were mixed and diluted with 500 μL of IP buffer (20 mM Tris, pH 7.5, 135mM NaCl, 1% Triton X-100, 10% glycerol, 1 mM EDTA, 1.5 mM MgCl₂)containing 6, 7, or 8 M urea. The reactions were precleared with 50 μLof Protein A Dynabeads (Invitrogen) for 15 minutes at 25° C. withrotation. The beads were then captured on a magnetic stand and thesupernatants were transferred to new tubes. Twenty μg of 1F11/3F5/Y102Lanti-linear, Apu3. A8 anti-K63, or an isotype control IgG was added toeach IP reaction and incubated at 25° C. with rotation for one hour.Next 100 μL of Protein A Dynabeads were added to each reaction and theIgGs were captured for 15 minutes at 25° C. with rotation. The beadswere then washed three times with 1 mL each of the corresponding bufferused in the IP (6, 7, or 8 M urea IP buffer) followed by two washes with1 mL each of PBS. During the final wash the beads were transferred tonew tubes to avoid eluting any proteins bound to the tube walls. Thebeads were resuspended in 30 μL of 1×LDS sample buffer (Invitrogen) withreducing agent (Invitrogen) and heated at 70° C. for 10 minutes to elutethe immunoprecipitated proteins. The beads were then captured on amagnetic stand and the supernatant was split in half and loaded induplicate onto two 4-12% Bis Tris NuPAGE 1.0 mm gels (Invitrogen). Aspositive and negative controls 1 μg each of purified linearpolyubiquitin 2-7 and K63-linked polyubiquitin 2-7 were also run on thegels. The gels were run in MES buffer (Invitrogen) and the western blotswere performed as described in the preceding paragraph. One blot wasprobed with 1F11/3F5/Y102L to detect linear chains and the other wasprobed with Apu3. A8 anti-K63 to detect K63-linked chains. In 6 M ureathere is a small amount of K63-linked chains pulled down by1F11/3F5/Y102L as seen in the previous IPs (see FIG. 18B). In 7 M urea1F11/3F5/Y102L behaves just like the isotype control and does not pulldown any K63-linked chains however it still retains the ability to IP asignificant amount of linear polyubiquitin when compared to what waspresent in the starting material. In 8 M urea, however the amount oflinear chains pulled down by 1F11/3F5/Y102L is dramatically reduced.Thus, 7 M urea is the most specific condition which strikes a balancebetween pulling down a significant amount of linear chains withoutbringing down K63-linked chains.

In the previous IPs the heavy chain of the IgGs used in the pull down iseluted from the beads and detected by the secondary antibody used in thewestern blots. This band can often obscure bands of the pull downmaterial. To make the blots cleaner and to be absolutely sure noK63-linked chains were being pulled down the IgGs were crosslinked tothe beads before the IPs. Twenty μg of either 1F11/3F5/Y102L or anisotype control IgG were incubated with 200 μL of Protein A Dynabeads inPBST for 30 minutes at 25° C. with rotation. The beads were captured ona magnetic stand and washed twice with 800 μL of conjugation buffer (20mM sodium phosphate pH 7.5, 150 mM NaCl). The IgG-coupled beads wereresuspended in 1 mL of 5 mM Bis(sulfosuccinimidyl)suberate (BS3) inconjugation buffer and incubated at 25° C. with rotation for 30 minutesto crosslink. While crosslinking the IgGs to the beads the IP reactionswere set up. Four μg each of linear polyubiquitin 2-7, K 11-linkedpolyubiquitin, K48-linked polyubiquitin 2-7, and K63-linkedpolyubiquitin 2-7 were mixed and diluted with 500 μL of IP buffer (20 mMTris, pH 7.5, 135 mM NaCl, 1% Triton X-100, 10% glycerol, 1 mM EDTA, 1.5mM MgCl₂) containing 0, 4, or 7 M urea. The mixtures were preclearedwith 50 μL of Protein A Dynabeads for 45 minutes at 25° C. withrotation. After 30 minutes the crosslinking reaction was quenched by theaddition of 48 μL of 1 M Tris pH 7.5 and incubation at 25° C. withrotation for 15 minutes. The crosslinked beads were washed three timeswith 800 μL of IP buffer containing the corresponding amount of urea tobe used in the IP (i.e. 0, 4, or 7 M urea). After washing theIgG-crosslinked beads were resuspended in the precleared IP reactionsand incubated at 25° C. with rotation for one hour. The IgG-crosslinkedbeads were then washed three times with 1 mL of IP buffer containing thecorresponding amount of urea followed by two washes with 1 mL of PBS.During the final wash the beads were transferred to new tubes to avoideluting any proteins bound to the tube walls. The beads were resuspendedin 50 μL of 1×LDS sample buffer (Invitrogen) with reducing agent(Invitrogen) and heated at 70° C. for 10 minutes to elute theimmunoprecipitated proteins. The beads were then captured on a magneticstand and the supernatant was split and loaded in quadruplicate onto4-12% Bis Tris NuPAGE 1.0 mm gels (Invitrogen). As positive and negativecontrols 1 μg each of purified linear polyubiquitin 2-7, K11-linkedpolyubiquitin, K48-linked polyubiquitin and K63-linked polyubiquitin 2-7were also run on the gels. The gels were run in MES buffer (Invitrogen)and the western blots were performed as described above in this example.One blot was probed with 1F11/3F5/Y102L to detect linear chains, oneblot was probed with 2A3/2E6 anti-K11 to detect K11-linked chains, oneblot was probed with Apu2.07 anti-K48 to detect K48-linked chains, andone blot was probed with Apu3. A8 anti-K63 to detect K63-linked chains.When comparing the amount of material in the IPs with the amount ofmaterial present in the starting inputs, overall much less linearpolyubiquitin was pulled down in each IP with 1F11/3F5/Y102L compared tothe IPs done with free IgG that was subsequently captured on the beadsin the previous paragraphs (see FIG. 19 ). This could be due to the factthat 1F11/3F5/Y102L is a member of the human IgG1 VH3 subgroup whichcontains a second Protein A binding site in the heavy chain variabledomain, in addition to the usual binding site in the Fc domain.Therefore, precoupling and crosslinking of the IgGs to Protein A beadsbefore antigen binding could diminish the binding capacity of thisantibody. Under these precoupling and crosslinking conditions 4 M ureais the most specific condition where 1F11/3F5/Y102L is able to pull downlinear chains without bringing down any K11-, K48-, or K63-linkedchains.

To see whether the reduction in linear chains pulled down was due to theadditional Protein A binding site in the heavy chain variable domain,the IPs were done using IgGs crosslinked to Protein G beads. Protein Galso has two binding sites on human IgG1 but both are in the constantdomains (CH1 and Fc). The IPs were repeated as described above exceptProtein G Dynabeads (Invitrogen) were used for precoupling andcrosslinking the IgGs. Much more linear polyubiquitin chains were pulleddown in each condition by 1F11/3F5/Y102L compared to the experimentswith the IgGs crosslinked to Protein A (see FIG. 20 ). Uponover-exposure of the anti-K63 blot at 4 M urea there is a small amountof K63-linked chains pulled down by 1F11/3F5/Y102L. Thus 7 M urea seemsto be the most specific condition when 1F11/3F5/Y102L is precoupled toProtein G beads however this comes at the expense of pulling down lesslinear polyubiquitin compared to when the IPs are done with free IgGthat is subsequently captured on beads (compare to FIG. 18B). It ispossible that precoupling and crosslinking of Protein G beads to the CH1domain may also diminish binding to linear polyubiquitin through sterichindrance of the neighboring VH domain, although less significantly thanprecoupling and crosslinking of Protein A beads to the VH domain.

The IPs using free IgG followed by subsequent capture with Protein Abeads were repeated using a mixture of polyubiquitin chains of differentlinkages as the substrate and were analyzed more extensively by westernblot and mass spectrometry. Two μg each of linear polyubiquitin 2-7(Boston Biochem), K11-linked polyubiquitin (Genentech), K48-linkedpolyubiquitin 2-7 (Boston Biochem), and K63-linked polyubiquitin 2-7(Boston Biochem) were mixed and diluted with 500 μL of IP buffer (20 mMTris, pH 7.5, 135 mM NaCl, 1% Triton X-100, 10% glycerol, 1 mM EDTA, 1.5mM MgCl₂) containing 0, 4, 5, 6 or 7 M urea. Each IP was done induplicate, one for western blots and the other for mass spectrometryanalysis. The reactions were precleared with 50 μL of Protein ADynabeads (Invitrogen) for 15 minutes at 25° C. with rotation. The beadswere then captured on a magnetic stand and the supernatants weretransferred to new tubes. Twenty μg of 1F11/3F5/Y102L anti-linear or anisotype control IgG was added to each IP reaction and incubated at 25°C. with rotation for one hour. Next 100 μL of Protein A Dynabeads wereadded to each reaction and the IgGs were captured for 15 minutes at 25°C. with rotation. The beads were then washed three times with 1 mL eachof the corresponding buffer used in the IP (0, 4, 5, 6, or 7 M urea IPbuffer) followed by two washes with 1 mL each of PBS. During the finalwash the beads were transferred to new tubes to avoid eluting anyproteins bound to the tube walls. The beads were resuspended in 50 μL of1×LDS sample buffer (Invitrogen) with reducing agent (Invitrogen) andheated at 70° C. for 10 minutes to elute the immunoprecipitatedproteins. The beads were then captured on a magnetic stand and thesupernatant was split and loaded in quadruplicate onto 4-12% Bis TrisNuPAGE 1.0 mm gels (Invitrogen) for western blots. As positive andnegative controls 500 ng each of purified linear polyubiquitin 2-7,K11-linked polyubiquitin, K48-linked polyubiquitin 2-7, and K63-linkedpolyubiquitin 2-7 were also run on the gels. The other set of IPs wererun on 4-12% Bis Tris NuPAGE 1.0 mm gels (Invitrogen) for massspectrometry AQUA analysis. The gels were run in MES buffer (Invitrogen)and the western blots were performed as described above in this example.The blots were probed with 1F11/3F5/Y102L anti-linear polyubiquitin,2A3/2E6 anti-K11-linked polyubiquitin, Apu2.07 anti-K48-linkedpolyubiquitin, and Apu3. A8 anti-K63 polyubiquitin antibodies (see FIG.21A). The other gels for mass spectrometry AQUA were stained withSimplyBlue Coomasie Safe stain (Invitrogen) (see FIG. 21B). In theabsence of urea, F11/3F5/Y102L is able to IP chains of all linkages (seeFIG. 21A). As the concentration of urea is increased in the IP buffer,the 1F11/3F5/Y102L IP becomes more specific. At 7 M urea no K11-linked,K48-linked, or K63-linked polyubiquitin is pulled down by the1F11/3F5/Y102L IgG and yet it is still able to pull down a significantamount of linear polyubiquitin relative to the starting input.

Regions B and C of the Coomassie stained gel were excised, subjected toin-gel tryptic digestion, and analyzed by mass spectrometry AQUA (seeFIG. 21B). Gel pieces were destained using 50 mM ammoniumbicarbonate/50% methanol and then desiccated with acetonitrile (ACN). Topermit effective uptake of trypsin, gel pieces were incubated on ice for2 hr with 20 ng/μL modified sequencing grade trypsin (Promega, MadisonWI) diluted in 50 mM ammonium bicarbonate/5% ACN. Digests were performedovernight at 37° C. and stopped by the addition of 50% ACN/5% formicacid (FA). Isotope labeled internal standard peptides (1 pmol) wereadded to each sample prior to two rounds of extraction (1st—50% ACN/5%FA 2nd—100% ACN). Extracted peptides were dried completely, andresuspended in 10% ACN/5% FA/0.01% H2O2 at least 30 minutes prior tomass spectrometric analysis. Samples were loaded directly onto a ThermoAQUASIL C18 column (2.1×150 mm) and separated using an Agilent 1200capillary LC at a flow rate of 200 μl/min over a 26 minute gradient of5% to 90% buffer B (98% ACN/0.1% FA). Mass spectrometric detection wasperformed on an ABI 4000 QTRAP using a segmented multiple reactionmonitoring (MRM) method for detecting both labeled and unlabeledpeptides covering the sequence of ubiquitin. Quantitation was performedby comparing peak areas between labeled and unlabeled versions of eachpeptide using ABI Multiquant 1.1 software. By measuring the abundancesof the -GG signature peptides corresponding to modification of the sevenlysines of ubiquitin and the amino terminus, 7M urea was confirmed to bethe most specific condition for immunoprecipitation of linearpolyubiquitin chains (see FIGS. 21C and 21D). In 7M urea the antibody isable to recover 67% of the linear chains present in the input fromregion B which contains the longer chains (tetraubiquitin throughheptaubiquitin) however it is less efficient at recovering the shortchains found in region C (diubiquitin and triubiquitin), with only 2% oflinear chains recovered (see FIG. 21E). This is likely due to avidityfrom the bivalent antibody and the multiple linkages found in longerchains.

E) Overexpression of LUBAC

The Linear Ubiquitin Assembly Complex (LUBAC) is an E3 ligase has beendemonstrated to assemble linear polyubiquitin chains (Kirisako, T. etal. (2006) EMBO J. 25:4877-4887). The open reading frames (ORFs) of twoof the members of this complex, Hoil-1L and Hoip, were synthesized (BlueHeron Biotechnology) and cloned into the pBI-CMV1 mammalian expressionvector (Clonetech) containing a bidirectional CMV promoter. Hoil-1L wascloned into multiple cloning site (MCS) 1 using restriction enzyme sitesMluI and EcoRV and Hoip was cloned into the MCS2 using restrictionenzyme sites EcoRI and PstI. The construct was verified by sequencingthe ORFs. The resulting construct, pBI-CMV1-Hoil1L-Hoip, or the emptyvector was transfected into 293T cells. 293T cells were split 1:20 into20 ten cm plates two days before transfection and grown at 37° C. in 5%CO2. On the day of transfection 150 μL of Lipofectamine 2000 was dilutedinto 2.5 mL of Opti-MEM media lacking serum for each plasmid to betransfected. Also 50 μg of either pBI-CMV1 empty vector orpBI-CMV1-Hoil1L-Hoip was diluted into 2.5 mL of Opti-MEM media lackingserum. These dilutions were incubated at 25° C. for five minutes. Thediluted Lipofectamine and the diluted DNA were combined, mixed gently byinversion, and incubated 25° C. for 30 minutes. 500 μL of theDNA/Lipofectamine mixture was then added to each ten cm plate (tenplates per plasmid). Forty-eight hours after transfection the media wascollected and the cells were scraped off the plates. The cells were spundown at 10,000 rpm for ten minutes at 4° C. The supernatants wereremoved and the cells were washed in 40 mL of cold PBS. The cells werespun down at 10,000 rpm for ten minutes at 4° C. The supernatants wereremoved and the cells were resuspended in 4 mL of lysis buffercontaining 8M urea, 50 mM Tris pH 7.5, 25 mM NaCl, 10 μL/mL HALTprotease and phosphatase inhibitors (Thermo Scientific), 5 mM EDTA, and2 mM NEM. Lysates were sonicated briefly to reduce viscosity and thenfrozen at −80° C. To determine whether Hoil-1L and Hoip wereoverexpressed and whether this leads to an increase in linearpolyubiquitin chain assembly, the lysates were analyzed by western blot.One μL of each lysate was mixed with LDS sample buffer containingreducing reagent and then loaded onto a 4-12% NuPAGE Bis Tris 1.0 mm gel(Invitrogen) and run in MES buffer (Invitrogen) in quadruplicate. Thegels were transferred individually at 30V for two hours in 1×NuPAGEtransfer buffer containing 10% methanol to 0.45 μm nitrocellulose. Themembranes were blocked in 5% milk in PBST for one hour at 25° C. withshaking and then incubated in the primary antibody. The first blot wasprobed with 1 μ/mL 1F11/3F5/Y102L anti-linear polyUb IgG in 5% milk inPBST. The second blot was probed with a 1:500 dilution of ananti-Hoil-1L/RBCK antibody (Abcam ab38540) in 5% milk in PBST. The thirdblot was probed with 1 μg/mL anti-Hoip/RNF31 antibody (Abcam ab85294) in5% milk in PBST. The fourth blot was probed with a 1:1000 dilution of ananti-β-tubulin antibody (Cell Signaling 9F3 #2128) in 5% milk in PBST.Blots one, two and three were incubated in their respective primaryantibodies for one hour at 25° C. with shaking. The anti-β-tubulin blotwas incubated overnight at 4° C. with rotation. Blots were then washedthree times in PBST0.05 and then incubated in a 1:10,000 dilution ofsecondary antibody in 5% milk in PBST for one hour at 25° C. withshaking. The anti-linear polyUb blot was probed with a goat anti-humanF(ab)′2-HRP secondary (Jackson Immunoresearch) and the other three blotswere probed with a goat anti-rabbit F(ab)′2-HRP secondary (JacksonImmunoresearch). The blots were then washed three times with PBST andthen once with PBS. The secondary antibody was detected using SuperSignal West Pico chemiluminescent substrate (Thermo Scientific) followedby exposure of the blots to film. The anti-p-tubulin blot demonstratesthat equal amounts of cells were used in the transfections and thatequivalent amounts of lysate were loaded on the gel (see FIG. 22A). Theanti-Hoil-1L and anti-Hoip blots show that when pBI-CMV1-Hoil1L-Hoip istransfected both Hoil-1L and Hoip are overexpressed relative toendogenous levels of the two proteins. Finally overexpression of Hoil-1Land Hoip leads to a dramatic increase in the levels of linear polyUbchains as expected. Given that these are detected by the 1F11/3F5/Y102LIgG indicates that this antibody can recognize endogenous, enzymaticallysynthesized, linear polyUb chains.

In addition to probing these lysates by western blot,immunoprecipitations of linear polyUb was also performed. 500 μL of theabove lysates were diluted to 7M urea with 71 μL of 50 mM Tris pH 7.5,25 mM NaCl. The lysates were then precleared with 200 μl of Protein ADynabeads (Invitrogen) in 7M urea, 50 mM Tris pH 7.5, 25 mM NaCl for onehour at 25° C. with rotation. The beads were captured with a magneticstand and the supernatants were transferred to new tubes. The preclearedlysates were then spun at 14,000 rpm for five minutes to pellet anyprecipitation. The supernatants were transferred to new tubes and 40 μgof 1F11/3F5/Y102L IgG or an isotype control IgG were added. Theimmunoprecipitations were then incubated overnight at 25° C. withrotation. The following day the IgGs were captured by the addition of200 μL of Protein A Dynabeads in 7M urea, 50 mM Tris pH 7.5, 25 mM NaClfor 15 minutes at 25° C. with rotation. The beads were captured on amagnetic stand and washed five times with 1 mL of 7M urea, 50 mM Tris pH7.5, 25 mM NaCl and then three times with 1 mL of PBS. After the finalwash the beads were transferred to new tubes to avoid eluting anyprotein sticking to the tube walls. The beads were then resuspended in30 μl of 1×LDS sample buffer with reducing agent (Invitrogen) and elutedat 70° C. for ten minutes. Two 4-12% NuPAGE Bis Tris 1.0 mm gels wererun: one for western blot and the other for Coomassie staining for massspec AQUA analysis. For western blot 10% of each IP was run along with0.1% of each lysate. For mass spec 90% of each IP was run along with 1%of each lysate. The western blot with 1F11/3F5/Y102L was done asdescribed in the above paragraph except the transfer done for one hour.The gel for mass spec AQUA analysis was stained with Simply Blue SafeStain (Invitrogen). The western blot shows that the 1F11/3F5/Y102L IgGcan immunoprecipitate linear polyUb chains from cells lysates (see FIG.22B). More chains are pulled down from the LUBAC overexpression lysatesthan from the vector alone consistent with the much higher levels oflinear chains present when LUBAC is overexpressed.

The high molecular weight regions of the gel indicated were excised (seeFIG. 22B) and subjected to mass spectrometry AQUA as described above inExample 4D. In the vector control samples no linear polyubiquitinlinkages were identified in the input samples (see Table 9). Themajority of the polyubiquitin chains identified were of the K48 and K63linkages.

TABLE 9 Vector Vector LUBAC LUBAC LUBAC Vector Isotype Linear OE OE OEInput IP IP Input Isotype IP Linear IP Linear 0 0 0 4808 0 598 K11 10850 0 700 0 12 K48 15680 2 7 12420 1 111 K63 5128 0 4 5075 0 76

Consistent with this, no linear polyubiquitin was pulled down in the IPwith either the anti-linear antibody or the isotype control antibody. InLUBAC over-expressed cells the levels of linear polyubiquitin linkagesreaches 4808 pmols, which is similar in abundance to K63 linkages (5075pmols). When the anti-linear antibody is used for IP it significantlyenriches for linear linkages with some additional K11, K48, and K63linkages being pulled down (see FIG. 22C). This is likely due to thepresence of mixed linkage chains or substrates modified with multiplehomogenous chains of different linkages since only 7 and 4 pmols of K48and K63 linkages, respectively, were pulled down by the anti-linearantibody from the vector control cells (see Table 9). Additionally, only1 pmol of K48 linkages and no K68 linkages were identified in the IPwith the isotype control antibody from LUBAC over-expressed cellsindicating that these linkages are simply not sticky. This demonstratesthat the anti-linear antibody is able to enrich for endogenous linearpolyubiquitin chains (see FIG. 22C).

To determine whether 1F11/3F5/Y102L is functional forimmunofluorescence, HeLa cells over-expressing Hoil-1L and Hoip werestained with the antibody. HeLa cells (5,000 cells/100 μl/well) wereseeded in a clear bottomed, black walled 96-well plate and grown for 24h. The cells were transfected with Hoil/Hoip plasmids for 18 hours usinglipofectamine 2000 according to manufacturer's protocols. The cells wererinsed with PBS, fixed with ice cold methanol at −20° C. for 10 minutes,permeabilized with PBS/0.1% Triton X-100 at room temperature for 5 min,then blocked with PBS/0.3% Triton X-100/5% BSA at room temperature for 1hour. The linear ubiquitin antibody (1 μg/ml) was incubated with orwithout poly-ubiquitin chains (5 μg/ml) at room temperature for 1 hourand then used to label cells at room temperature for 1 hour. After 6washes (10 min each) with PBS/0.05% Triton X-100, the cells were stainedwith DyLight488-conjugated donkey anti-human antibody (1:500, JacksonImmunoResearch Laboratories) at room temperature for 1 hour. The cellsthen were washed 6 times (10 minutes each) with PBS containing 0.05%Triton X-100, stained with Hoechst (1:10,000) at room temperature for 10min and washed with PBS. The plate was covered with black seals andimaged using ImageXpress Micro imaging system. Untransfected cells didnot show any signal when stained with 1F11/3F5/Y102L but cellsover-expressing Hoil-1L and Hoip demonstrated a cytoplasmic punctatestaining pattern (see FIG. 22D). This staining was specific for linearpolyubiquitin chains as it could be blocked by the addition ofrecombinant linear polyubiquitin.

Example 5—Structural Analysis of Fab Binding to Linear Diubiquitin

To better understand the interaction of the anti-linear Fab with linearpolyubiquitin the 1F11/3F5/Y102L Fab was co-crystallized with lineardiubiquitin. The Fab fragment of 1F11/3F5/Y102L was expressed in E. coliand purified over a Protein A column as described above in Example 1E.The Fab was further purified over a 5 mL SP HiTrap column (GEHealthcare) in 20 mM 2-(N-morpholino)ethanesulfonic acid (MES), pH 5.5with a 0-100% linear gradient of 20 mM MES, pH 5.5, 0.5 M NaCl.Fractions containing the Fab fragment were pooled and run over a 320 mLS75 sizing column (GE Healthcare) in 25 mM Tris, pH 7.5, 150 mM NaCl.Fractions containing the Fab fragment were pooled and concentrated to 22mg/mL.

A head-to-tail fusion of two ubiquitin subunits through a canonicalpeptide bond (linear diubiquitin) was also expressed in E. coli.BL21-Gold (Agilent Technologies) cells were transformed with a lineardiubiquitin expression plasmid constructed in a pET15b vector (Novagen).This construct has an N-terminal His6 tag (SEQ ID NO: 376) followed by athrombin cleavage site under the control of the T7 promoter and lacoperator. An overnight culture of BL21-Gold transformed with thepET15b-linear diubiquitin expression plasmid grown in LB media wasdiluted 50-fold into 1L of warmed Terrific Broth containing 1% glycerol,0.1 M MOPS, pH 7.3, and 50 μg/mL carbenicilin. The culture was grown at37° C. with shaking at 250 rpms in a 2.5 L ultra-yield flask (Thomson)to an OD₆₀₀=1.64. Expression was induced by adding 0.5 mM IPTG and theculture was grown overnight at 16° C. with shaking at 250 rpms. Next daythe cells were pelleted by spinning at 8K rpm for 10 minutes and thepellets were frozen in liquid nitrogen. Cells were resuspended and lysedin 40 mM Tris, pH 8.0, 0.3 M NaCl, and Complete EDTA-free proteaseinhibitor tablets (Roche) by microfluidizing three times. Cell debriswas pelleted by spinning at 10K rpm for 1 hour and the supernatant wasfiltered through a 0.45 μM low protein-binding filter. The His6-diUb(‘His₆’ disclosed as SEQ ID NO: 376) was purified over a 5 mL Ni-NTAagarose (Qiagen) column. The column was washed with 12 column volumes ofBuffer A (20 mM Tris, pH 8.0, 1 M NaCl, 20 mM imidazole) and eluted withfour column volumes of Buffer B (20 mM Tris, pH 8.0, 1 M NaCl, 250 mMimidazole). The expression of the diubiquitin was high such that itexceeded the binding capacity of the column and some diubiquitin waseluted in the wash with Buffer A. Thus the wash material was run over asecond 5 mL Ni-NTA agarose column and purified as described above. TheHis6 tag (SEQ ID NO: 376) was removed by cleaving with 1800 units ofThrombin (GE Healthcare) at 4° C. for 3 days while dialyzing into 25 mMTris, pH 8.0, 150 mM NaCl, 2 mM CaCl₂) using a 3500 MWCO dialysis tubing(Spectrum Medical). The dialyzed and cleaved material was split in halfand the free diubiquitin was separated from the His6 tag (SEQ ID NO:376) using two 5 mL Ni-NTA agarose columns. The flow through and allwashes were collected. The columns were washed with four column volumesof Buffer C (25 mM Tris, pH 8.0, 0.5 M NaCl), four column volumes ofBuffer D (25 mM Tris, pH 8.0, 0.5 M NaCl, 20 mM imidazole), and then onecolumn volume of Buffer E (25 mM Tris, pH 8.0, 0.5 M NaCl, 250 mMimidazole). The presence of detagged diubiquitin was monitored byrunning samples from the flow through and each wash on an 18% Novextris-glycine gel (Invitrogen) and staining with Simply Blue Safe Stain(Invitrogen). The majority of the detagged diubiquitin was present inthe flow through, wash with Buffer C, and wash with Buffer D. These werepooled, concentrated and purified further over a 320 mL S75 sizingcolumn (GE Healthcare) in 25 mM Tris, pH 7.5, 150 mM NaCl. Fractionscontaining the diubiquitin were pooled and concentrated to 15.3 mg/mL.

The Fab/diubiquitin complex was set up using a three-fold molar excessof linear diubiquitin to Fab and was incubated at 4° C. overnight. Thecomplex was then purified over a 320 mL S75 sizing column (GEHealthcare) in 25 mM Tris, pH 7.5, 150 mM NaCl. Fractions containing thecomplex were pooled and concentrated to 20 mg/mL.

Crystals were grown using the sitting drop vapor diffusion method indrops containing 0.2 μL of 1F11/3F5/Y102L Fab/linear diubiquitin complex(20 mg/mL complex in 25 mM Tris, pH 7.5, 150 mM NaCl) and 0.2 μL ofmother liquor (20% isopropanol, 0.1 M MES, pH 6.0, 20% polyethyleneglycol (PEG) 2K monomethylether (MME)). Initial crystals grew underthese conditions at 19° C. over 27 days. Crystals were optimized insitting drops using a microbridge with 2 μL of 1F11/3F5/Y102L Fab/lineardiubiquitin complex (20 mg/mL complex in 25 mM Tris, pH 7.5, 150 mMNaCl) and 3 μL of mother liquor (18% isopropanol, 0.09 M MES, pH 6.0,19.8% PEG 2K MME, 10 mM sodium bromide). Crystals grew at 19° C. overfive days and could be manipulated to obtain single, diffractingcrystals. Crystals were cryoprotected using 20% isopropanol, 0.1 M MES,pH 6.0, 20% PEG 2K MME with an additional 25-30% PEG 2K MME.Crystallographic data was collected at Berkeley Advanced Light Sourcebeamline 5.0.2 and was processed using HKL2000. Crystals belonged to theP1 space group with unit cell dimensions a=53 Å, b=60 Å, c=96 Å, α=87°,β=77°, and γ=72°, with two complexes in the asymmetric unit. Thestructure was solved by molecular replacement using the program Phaserand the coordinates of a variant of the humanized 4D5 Fab fragment (PDBcode for 4D5: 1FVE) and of human monoubiquitin (PDB code: 1UBQ). Modelbuilding was carried out in Coot and the structure was refined usingPhenix. The resolution of the structure is 2.43 Å and the complex hasbeen refined to an R of 22.8% and Rfree of 25.0% (see FIG. 23 . PanelA).

Analysis of the structure indicates that the majority of the binding todiubiquitin is mediated through contacts with the heavy chain CDRs.Between the heavy chain and diubiquitin there is 785 Å² of buriedsurface area, in contrast to no buried surface area between the lightchain and diubiquitin. The structural epitope and paratope is defined asresidues which bury at least 25% of their solvent accessible surfacearea upon binding (see FIG. 23 Panels B and C) and/or have at least oneatom within 4.5 Å of the interacting chain (see Tables 10 and 11).

TABLE 10 Residues with at least 25% of solvent accessible surface areaburied at the interface (Table 10 discloses residues 52-57 and 96-100 asSEQ ID NOS 377 and 378, respectively.) Fab HC (chain C) DiUb (chain A)N31 L8 Nterm Ub T32 T9 Nterm Ub Y33 E34 Nterm Ub T52 I36 Nterm Ub P53(Kabat# P52a) P37 Nterm Ub S54 (S53) Q40 Nterm Ub S55 (S54) R42 Nterm UbG56 (G55) V70 Nterm Ub Q57 (Q56) L71 Nterm Ub T74 (T73) L73 Nterm Ub T96(T95) R74 Nterm Ub W97 (W96) G75 Nterm Ub L98 (L97) M1 Cterm Ub L99(L98) P19 Cterm Ub R100 (R99) S57 Cterm Ub D58 Cterm Ub N60 Cterm Ub I61Cterm Ub Q62 Cterm Ub K63 Cterm Ub

TABLE 11 Residues with at least 1 atom within 4.5Å of Fab or diUb (Table11 discloses residues 52-57, 96-100, 31-37, 70-76 and 60-63 as SEQ IDNOS 377-381, respectively.) Fab HC Fab LC DiUb (chain C) (chain B)(chain A) N31 Y49 L8 Nterm Ub T32 S50 T9 Nterm Ub Y33 F53 Q31 Nterm UbT52 Y55 D32 Nterm Ub P53 S56 K33 Nterm Ub (Kabat#P52a) S54 (S53) E34Nterm Ub S55 (S54) G35 Nterm Ub G56 (G55) I36 Nterm Ub Q57 (Q56) P37Nterm Ub T74 (T73) Q40 Nterm Ub T96 (T95) R42 Nterm Ub W97 (W96) V70Nterm Ub L98 (L97) L71 Nterm Ub L99 (L98) R72 Nterm Ub R100 (R99) L73Nterm Ub R74 Nterm Ub G75 Nterm Ub G76 Nterm Ub M1 Cterm Ub P19 Cterm UbS57 Cterm Ub D58 Cterm Ub N60 Cterm Ub I61 Cterm Ub Q62 Cterm Ub K63Cterm Ub

There are 15 heavy chain residues and only five light chain residueswhich make up the Fab paratope. The diubiquitin epitope consists of 18residues from the distal Ub (C-terminus involved in the linkage) andeight residues from the proximal ubiquitin (N-terminus involved in thelinkage). The contact interface also consists of nine hydrogen bondsbetween the heavy chain and diubiquitin and three hydrogen bonds betweenthe light chain and diubiquitin (see Table 12). Rather than makingexclusive contact with the linear linkage itself, the specificityappears to be derived from multiple interactions with surface residuesfrom both the proximal and distal ubiquitins.

TABLE 12 Summary of H-bonds between Fab and diUb diUb (chain A) Fab LC(chain B) Nterm Ub E34 O Y49 OH Nterm Ub E34 OE2 Y49 OH Nterm Ub G35 OS50 OG diUb (chain A) Fab HC (chain C) Nterm Ub Q40 NE2 R100 N (Kabat#R99) Nterm Ub R42 NH1 N31 ND2 Nterm Ub R74 N Y33 OH Nterm Ub R74 O R100NH2 (R99) Nterm Ub G75 O Q57 NE2 (Q56) Nterm Ub G76 O Q57 NE2 (Q56)Cterm Ub Q62 NE2 P53 O (P52a) Cterm Ub Q62 OE2 G56 N (G55) Cterm Ub K63N S55 O (S54)

Despite free linear polyubiquitin and free K63-linked polyubiquitinchains adopting similar structures due to Lys63 and the freeamino-terminus situated only ˜6 Å apart in the monoubiquitin structure(PDB code 1UBQ), this antibody preferentially binds linear chains. Thisspecificity is achieved through the dual recognition of the relativeorientations of both the proximal and distal ubiquitin subunits.Recognition of the proximal ubiquitin is achieved through interaction ofCDR H2 residues with the 60s loop of the proximal subunit. Inparticular, two hydrogen bonds are made with the side-chain of Gln62 ofthe proximal ubiquitin, both involving residues of CDR H2. One isbetween the main-chain carbonyl oxygen of Pro52a and the side-chainamine of Gln62. The other is between the main-chain amide of Gly55 andthe side-chain carbonyl of Gln62. There is also a third hydrogen bondbetween the main-chain carbonyl of Ser54 and the main-chain amide ofLys63. In addition to these three hydrogen bonds, numerous Van der Waalsinteractions also occur between CDR H2 and the proximal ubiquitin.Although the distal ubiquitin of K63-linked diubiquitin couldtheoretically bind in the same orientation to the antibody, the proximalubiquitin would be rotated slightly due to the ˜6 Å difference inposition of Met1 and Lys63. This rotation would likely disrupt thehydrogen bonds and Van der Waals interactions between CDR H2 and the60's loop. Therefore specificity is encoded by recognition of therelative spatial orientations of the proximal and distal ubiquitinsresulting from the linear linkage.

The structure also illustrates why the particular mutations selected inthe affinity maturation help increase affinity. In CDR H2 the mutationthat made the most significant improvement in sensitivity in westernblots was S56Q (see FIG. 13 , clone 3F5). The structure shows that Gln56makes two hydrogen bonds: one with the carbonyl oxygen of Gly75 of thedistal ubiquitin and the second with the carbonyl oxygen of Gly76 of thedistal ubiquitin, which participates in the linear linkage between thedistal and proximal ubiquitins (see FIG. 24 , Panel A). The shorter sidechain of Ser likely would not reach these residues to make the hydrogenbonds. The mutation which made the second largest improvement insensitivity in western blots was S52K in CDR L2 (see FIG. 13 , clone1F11). Lys52 is placed in proximity to Asp32 found at thecarboxy-terminal end of the alpha helix of the distal ubiquitin.Although Lys52 is too far away to make a salt bridge with the side chainof Asp32 it may make a favorable electrostatic interaction given that itis also oriented towards the negative end of the helix dipole (see FIG.24 , Panel B). The third mutation which improved sensitivity of theantibody was Y102L in CDR H3 (see FIG. 13 , clone Y102L). Although thisresidue does not contact the diubiquitin it may help stabilize afavorable conformation of CDR H3 for improved binding. Leu102intercalates between Val2 and Leu4 of framework 1 of the heavy chain(see FIG. 24 , Panel C). The more hydrophobic side chain of Leu couldprovide a more favorable interaction with Val2 and Leu4 than Tyr andmight help position the rest of CDR H3 to make contacts with thediubiquitin.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

1-55. (canceled)
 56. A method of determining the function and/oractivity of C- to N-terminal-linked polyubiquitin in a cell or samplecomprising contacting the cell or sample with at least one antibody andassessing the effect of said contacting step on the cell or sample,wherein the at least one antibody comprises: a) the hypervariable(HVR)-L1 sequence of SEQ ID NO: 1, the HVR-L2 sequence of SEQ ID NO: 58,the HVR-L3 sequence of SEQ ID NO: 3, the HVR-H1 sequence of SEQ ID NO:7, the HVR-H2 sequence of SEQ ID NO: 82, and the HVR-H3 sequence of SEQID NO: 9: or b) the HVR-L1 sequence of SEQ ID NO: 1, the HVR-L2 sequenceselected from SEQ ID NO: 2, 58 and 60, the HVR-L3 sequence selected fromSEQ ID NO: 3, 65 and 72, the HVR-H1 sequence of SEQ ID NO: 7 or 80, theHVR-H2 sequence of SEQ ID NO: 8 or 82, and the HVR-H3 sequence of SEQ IDNO: 9 or 87; or c) the HVR-L1 sequence of SEQ ID NO: 1, the HVR-L2sequence of SEQ ID NO: 58, the HVR-L3 sequence of SEQ ID NO: 3, theHVR-H1 sequence of SEQ ID NO: 7, the HVR-H2 sequence of SEQ ID NO: 8,and the HVR-H3 sequence of SEQ ID NO: 9; or d) the HVR-L1 sequence ofSEQ ID NO: 1, the HVR-L2 sequence of SEQ ID NO: 2, the HVR-L3 sequenceof SEQ ID NO: 3, the HVR-H1 sequence of SEQ ID NO: 7, the HVR-H2sequence of SEQ ID NO: 82, and the HVR-H3 sequence of SEQ ID NO: 9; ore) the HVR-L1 sequence of SEQ ID NO: 1, the HVR-L2 sequence of SEQ IDNO: 2, the HVR-L3 sequence of SEQ ID NO: 3, the HVR-H1 sequence of SEQID NO: 10, the HVR-H2 sequence of SEQ ID NO: 11, and the HVR-H3 sequenceof SEQ ID NO: 12; or f) the HVR-L1 sequence of SEQ ID NO: 19, the HVR-L2sequence of SEQ ID NO: 2, the HVR-L3 sequence of SEQ ID NO: 3, theHVR-H1 sequence of SEQ ID NO: 10, the HVR-H2 sequence of SEQ ID NO: 23,and the HVR-H3 sequence of SEQ ID NO: 12; or g) the HVR-L1 sequence ofSEQ ID NO: 1, the HVR-L2 sequence of SEQ ID NO: 2, the HVR-L3 sequenceof SEQ ID NO: 20, the HVR-H1 sequence of SEQ ID NO: 10, the HVR-H2sequence of SEQ ID NO: 11, and the HVR-H3 sequence of SEQ ID NO: 12; orh) the HVR-L1 sequence of SEQ ID NO: 1, the HVR-L2 sequence of SEQ IDNO: 2, the HVR-L3 sequence of SEQ ID NO: 21, the HVR-H1 sequence of SEQID NO: 22, the HVR-H2 sequence of SEQ ID NO: 24, and the HVR-H3 sequenceof SEQ ID NO: 12; or i) the HVR-L1 sequence of SEQ ID NO: 1, the HVR-L2sequence of SEQ ID NO: 2, the HVR-L3 sequence of SEQ ID NO: 3, theHVR-H1 sequence of SEQ ID NO: 7, the HVR-H2 sequence of SEQ ID NO: 8,and the HVR-H3 sequence of SEQ ID NO: 9; or j) the HVR-L1 sequence ofSEQ ID NO: 4, the HVR-L2 sequence of amino acids 50-56 of SEQ ID NO: 27,the HVR-L3 sequence of SEQ ID NO: 5, the HVR-H1 sequence of SEQ ID NO:13, the HVR-H2 sequence of amino acids 52-62 of SEQ ID NO: 31, and theHVR-H3 sequence of SEQ ID NO: 15: or the HVR-L1 sequence of SEQ ID NO:4, the HVR-L2 sequence of amino acids 50-56 of SEQ ID NO: 28, the HVR-L3sequence of SEQ ID NO: 6, the HVR-H1 sequence of SEQ ID NO: 16, theHVR-H2 sequence of amino acids 52-62 of SEQ ID NO: 32, and the HVR-H3sequence of SEQ ID NO: 18; or k) the HVR-L1 sequence of SEQ ID NO: 1,50, 52-57, or 199-218, the HVR-L2 sequence of SEQ ID NO: 2, the HVR-L3sequence of SEQ ID NO: 3, the HVR-H1 sequence of SEQ ID NO: 7, theHVR-H2 sequence of SEQ ID NO: 8, and the HVR-H3 sequence of SEQ ID NO:9; or l) the HVR-L1 sequence of SEQ ID NO: 1, the HVR-L2 sequence of SEQID NO: 58-62 or 219-239, the HVR-L3 sequence of SEQ ID NO: 3, the HVR-H1sequence of SEQ ID NO: 7, the HVR-H2 sequence of SEQ ID NO: 8, and theHVR-H3 sequence of SEQ ID NO: 9; or m) the HVR-L1 sequence of SEQ ID NO:1, the HVR-L2 sequence of SEQ ID NO: 2, the HVR-L3 sequence of SEQ IDNO: 6, 64-72, or 240-254, the HVR-H1 sequence of SEQ ID NO: 7, theHVR-H2 sequence of SEQ ID NO: 8, and the HVR-H3 sequence of SEQ ID NO:9; or n) the HVR-L1 sequence of SEQ ID NO: 1, the HVR-L2 sequence of SEQID NO: 2, the HVR-L3 sequence of SEQ ID NO: 3, the HVR-H1 sequence ofSEQ ID NO: 73-81 or 255-260, the HVR-H2 sequence of SEQ ID NO: 8, andthe HVR-H3 sequence of SEQ ID NO: 9, or o) the HVR-L1 sequence of SEQ IDNO: 1, the HVR-L2 sequence of SEQ ID NO: 2, the HVR-L3 sequence of SEQID NO: 3, the HVR-H1 sequence of SEQ ID NO: 7, the HVR-H2 sequence ofSEQ ID NO: 17, 82-86, or 261-264, and the HVR-H3 sequence of SEQ ID NO:9; or p) the HVR-L1 sequence of SEQ ID NO: 1, the HVR-L2 sequence of SEQID NO: 2, the HVR-L3 sequence of SEQ ID NO: 3, the HVR-H1 sequence ofSEQ ID NO: 7, the HVR-H2 sequence of SEQ ID NO: 8, and the HVR-H3sequence of SEQ ID NO: 265-282.
 57. The method of claim 56, wherein theat least one antibody comprises a HVR-L1 comprising the sequence of SEQID NO: 1, a HVR-L2 sequence comprising the sequence of SEQ ID NO: 58, aHVR-L3 sequence comprising the sequence of SEQ ID NO: 3, a HVR-H1sequence comprising the sequence of SEQ ID NO: 7, a HVR-H2 sequencecomprising the sequence of SEQ ID NO: 82, and a HVR-H3 sequencecomprising the sequence of SEQ ID NO:
 9. 58. The method of claim 57,wherein the first amino acid after the C-terminus of HVR-H3 is aleucine.
 59. The method of claim 56, wherein the at least one antibodycomprises a HVR-L1 comprising the sequence of SEQ ID NO: 1, a HVR-L2sequence comprising the sequence of SEQ ID NO: 58, a HVR-L3 sequencecomprising the sequence of SEQ ID NO: 72, a HVR-H1 sequence comprisingthe sequence of SEQ ID NO: 7, a HVR-H2 sequence comprising the sequenceof SEQ ID NO: 82, and a HVR-H3 sequence comprising the sequence of SEQID NO:
 9. 60. The method of claim 56, wherein the at least one antibodycomprises a HVR-L1 comprising the sequence of SEQ ID NO: 1, a HVR-L2sequence comprising the sequence of SEQ ID NO: 2, a HVR-L3 sequencecomprising the sequence of SEQ ID NO: 65, a HVR-H1 sequence comprisingthe sequence of SEQ ID NO: 7, a HVR-H2 sequence comprising the sequenceof SEQ ID NO: 82, and a HVR-H3 sequence comprising the sequence of SEQID NO:
 9. 61. The method of claim 56, wherein the at least one antibodycomprises a HVR-L1 comprising the sequence of SEQ ID NO: 1, a HVR-L2sequence comprising the sequence of SEQ ID NO: 2, a HVR-L3 sequencecomprising the sequence of SEQ ID NO: 65 or 72, a HVR-H1 sequencecomprising the sequence of SEQ ID NO: 7, a HVR-H2 sequence comprisingthe sequence of SEQ ID NO: 82, and a HVR-H3 sequence comprising thesequence of SEQ ID NO:
 9. 62. The method of claim 56, wherein the atleast one antibody comprises a HVR-L1 comprising the sequence of SEQ IDNO: 1, a HVR-L2 sequence comprising the sequence of SEQ ID NO: 58, aHVR-L3 sequence comprising the sequence of SEQ ID NO: 65, a HVR-H1sequence comprising the sequence of SEQ ID NO: 7, a HVR-H2 sequencecomprising the sequence of SEQ ID NO: 82, and a HVR-H3 sequencecomprising the sequence of SEQ ID NO:
 9. 63. The method of claim 56,wherein the at least one antibody comprises a light chain comprising thesequence of SEQ ID NO: 94 and a heavy chain comprising the sequence ofSEQ ID NO:
 95. 64. The method of claim 56, wherein the at least oneantibody does not specifically bind monoubiquitin and/or does not bind asecond polyubiquitin comprising a lysine linkage.
 65. The method ofclaim 56, wherein the at least one antibody is a monoclonal antibody, ahumanized antibody, a chimeric antibody, or an antibody fragment thatbinds C- to N-terminal-linked polyubiquitin.
 66. The method of claim 56,wherein the at least one antibody comprises the HVR-L1 sequence of SEQID NO: 1, the HVR-L2 sequence of SEQ ID NO: 58, the HVR-L3 sequence ofSEQ ID NO: 3, the HVR-H1 sequence of SEQ ID NO: 7, the HVR-H2 sequenceof SEQ ID NO: 8, and the HVR-H3 sequence of SEQ ID NO:
 9. 67. The methodof claim 56, wherein the at least one antibody comprises the HVR-L1sequence of SEQ ID NO: 1, the HVR-L2 sequence of SEQ ID NO: 2, theHVR-L3 sequence of SEQ ID NO: 3, the HVR-H1 sequence of SEQ ID NO: 7,the HVR-H2 sequence of SEQ ID NO: 82, and the HVR-H3 sequence of SEQ IDNO:
 9. 68. The method of claim 56, wherein the at least one antibodycomprises the HVR-L1 sequence of SEQ ID NO: 1, the HVR-L2 sequence ofSEQ ID NO: 2, the HVR-L3 sequence of SEQ ID NO: 3, the HVR-H1 sequenceof SEQ ID NO: 10, the HVR-H2 sequence of SEQ ID NO: 11, and the HVR-H3sequence of SEQ ID NO:
 12. 69. The method of claim 56, wherein the atleast one antibody comprises the HVR-L1 sequence of SEQ ID NO: 19, theHVR-L2 sequence of SEQ ID NO: 2, the HVR-L3 sequence of SEQ ID NO: 3,the HVR-H1 sequence of SEQ ID NO: 10, the HVR-H2 sequence of SEQ ID NO:23, and the HVR-H3 sequence of SEQ ID NO:
 12. 70. The method of claim56, wherein the at least one antibody comprises the HVR-L1 sequence ofSEQ ID NO: 1, the HVR-L2 sequence of SEQ ID NO: 2, the HVR-L3 sequenceof SEQ ID NO: 20, the HVR-H1 sequence of SEQ ID NO: 10, the HVR-H2sequence of SEQ ID NO: 11, and the HVR-H3 sequence of SEQ ID NO:
 12. 71.The method of claim 56, wherein the at least one antibody comprises theHVR-L1 sequence of SEQ ID NO: 1, the HVR-L2 sequence of SEQ ID NO: 2,the HVR-L3 sequence of SEQ ID NO: 21, the HVR-H1 sequence of SEQ ID NO:22, the HVR-H2 sequence of SEQ ID NO: 24, and the HVR-H3 sequence of SEQID NO:
 12. 72. The method of claim 56, wherein the at least one antibodycomprises the HVR-L1 sequence of SEQ ID NO: 1, the HVR-L2 sequence ofSEQ ID NO: 2, the HVR-L3 sequence of SEQ ID NO: 3, the HVR-H1 sequenceof SEQ ID NO: 7, the HVR-H2 sequence of SEQ ID NO: 8, and the HVR-H3sequence of SEQ ID NO: 9.